Engineered CRISPR-Cas9 Nucleases with Altered PAM Specificity

ABSTRACT

Engineered CRISPR-Cas9 nucleases with altered and improved PAM specificities and their use in genomic engineering, epigenomic engineering, and genome targeting.

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No.15/208,228, filed Jul. 12, 2016, which is a divisional application ofU.S. patent application Ser. No. 15/060,424, filed Mar. 3, 2016, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.62/127,634, filed on Mar. 3, 2015; 62/165,517, filed on May 22, 2015;62/239,737, filed on Oct. 9, 2015; and 62/258,402, filed on Nov. 20,2015. This application is also a continuation of U.S. patent applicationSer. No. 15/664,873, filed Jul. 31, 2017, which is a continuationapplication of U.S. patent application Ser. No. 15/208,461, filed Jul.12, 2016, now U.S. Pat. No. 9,752,132, which is a continuationapplication of U.S. patent application Ser. No. 15/060,448, filed Mar.3, 2016, which claims the benefit of U.S. Provisional Patent ApplicationSer. No. 62/127,634, filed on Mar. 3, 2015; 62/165,517, filed on May 22,2015; 62/239,737, filed on Oct. 9, 2015; and 62/258,402, filed on Nov.20, 2015. The entire contents of the foregoing are hereby incorporatedby reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos. DP1GM105378, NIH R01 GM107427, and R01 GM088040 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

TECHNICAL FIELD

The invention relates, at least in part, to engineered ClusteredRegularly Interspaced Short Palindromic Repeats(CRISPRs)/CRISPR-associated protein 9 (Cas9) nucleases with altered andimproved Protospacer Adjacent Motif (PAM) specificities and their use ingenomic engineering, epigenomic engineering, and genome targeting.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 12, 2016, isnamed 29539-0163003_SL.txt and is 925,050 bytes in size.

BACKGROUND

CRISPR-Cas9 nucleases enable efficient, customizable genome editing in awide variety of organisms and cell types (Sander & Joung, Nat Biotechnol32, 347-355 (2014); Hsu et al., Cell 157, 1262-1278 (2014); Doudna &Charpentier, Science 346, 1258096 (2014); Barrangou & May, Expert OpinBiol Ther 15, 311-314 (2015)). Target site recognition by Cas9 isdirected by two short RNAs known as the crRNA and tracrRNA (Deltcheva etal., Nature 471, 602-607 (2011); Jinek et al., Science 337, 816-821(2012)), which can be fused into a chimeric single guide RNA (sgRNA)(Jinek et al., Science 337, 816-821 (2012); Jinek et al., Elife 2,e00471 (2013); Mali et al., Science 339, 823-826 (2013); Cong et al.,Science 339, 819-823 (2013)). The 5′ end of the sgRNA (derived from thecrRNA) can base pair with the target DNA site, thereby permittingstraightforward re-programming of site-specific cleavage by theCas9/sgRNA complex (Jinek et al., Science 337, 816-821 (2012)). However,Cas9 must also recognize a specific protospacer adjacent motif (PAM)that lies proximal to the DNA that base pairs with the sgRNA (Mojica etal., Microbiology 155, 733-740 (2009); Shah et al., RNA Biol 10, 891-899(2013); Jinek et al., Science 337, 816-821 (2012); Sapranauskas et al,Nucleic Acids Res 39, 9275-9282 (2011); Horvath et al., J Bacteriol 190,1401-1412 (2008)), a requirement that is needed to initiatesequence-specific recognition (Sternberg et al., Nature 507, 62-67(2014)) but that can also constrain the targeting range of thesenucleases for genome editing. The broadly used Streptococcus pyogenesCas9 (SpCas9) recognizes a short NGG PAM (Jinek et al., Science 337,816-821 (2012); Jiang et al., Nat Biotechnol 31, 233-239 (2013)), whichoccurs once in every 8 bps of random DNA sequence. By contrast, otherCas9 orthologues characterized to date can recognize longer PAMs(Horvath et al., J Bacteriol 190, 1401-1412 (2008); Fonfara et al.,Nucleic Acids Res 42, 2577-2590 (2014); Esvelt et al., Nat Methods 10,1116-1121 (2013); Ran et al., Nature 520, 186-191 (2015); Zhang et al.,Mol Cell 50, 488-503 (2013)). For example, Staphylococcus aureus Cas9(SaCas9), one of several smaller Cas9 orthologues that are better suitedfor viral delivery (Horvath et al., J Bacteriol 190, 1401-1412 (2008);Ran et al., Nature 520, 186-191 (2015); Zhang et al., Mol Cell 50,488-503 (2013)), recognizes a longer NNGRRT (SEQ ID NO:46) PAM that isexpected to occur once in every 32 bps of random DNA. Broadening thetargeting range of Cas9 orthologues is important for variousapplications including the modification of small genetic elements (e.g.,transcription factor binding sites (Canver et al. Nature;527(7577):192-7 (2015); Vierstra et al., Nat Methods. 12(10):927-30(2015)) or performing allele-specific alterations by positioningsequence differences within the PAM (Courtney, D. G. et al. Gene Ther.23(1):108-12 (2015).

SUMMARY

As described herein, the commonly used Streptococcus pyogenes Cas9(SpCas9) as well as the Staphylococcus aureus Cas9 (SaCas9) wereengineered to recognize novel PAM sequences using structuralinformation, bacterial selection-based directed evolution, andcombinatorial design. These altered PAM specificity variants enablerobust editing of endogenous gene sites in zebrafish and human cellsthat cannot be efficiently targeted by wild-type SpCas9 or SaCas9. Inaddition, we identified and characterized another SpCas9 variant thatexhibits improved PAM specificity in human cells, possessing reducedactivity on sites with non-canonical NAG and NGA PAMs. Furthermore, wefound that two smaller-size Cas9 orthologues with completely differentPAM specificities, Streptococcus thermophilus Cas9 (St1Cas9) andStaphylococcus aureus Cas9 (SaCas9), function efficiently in ourbacterial selection system and in human cells, suggesting that ourengineering strategies could be extended to Cas9s from other species.Our findings provide broadly useful SpCas9 and SaCas9 variants, referredto collectively herein as “variants” or “the variants”.

In a first aspect, the invention provides isolated Streptococcuspyogenes Cas9 (SpCas9) proteins with mutations at one or more of thefollowing positions: G1104, S1109, L1111, D1135, S1136, G1218, N1317,R1335, T1337, e.g., comprising a sequence that is at least 80% identicalto the amino acid sequence of SEQ ID NO:1. In some embodiments, thevariant SpCas9 proteins comprise one or more of the following mutations:G1104K; S1109T; L1111H; D1135V; D1135E; D1135N; D1135Y; S1136N; G1218R;N1317K; R1335E; R1335Q; and T1337R. In some embodiments, the variantSpCas9 proteins comprise the following mutations: D1135;D1135V/R1335Q/T1337R (VQR variant); D1135E/R1335Q/T1337R (EQR variant);D1135V/G1218/R1335Q/T1337R (VRQR variant); D1135N/G1218R/R1335Q/T1337R(NRQR variant); D1135Y/G1218R/R1335Q/T1337R (YRQR variant);G1104K/D1135V/G1218R/R1335Q/T1337R (KVRQR variant);S1109T/D1135V/G1218R/R1335Q/T1337R (TVRQR variant);L1111H/D1135V/G1218R/R1335Q/T1337R (HVRQR variant);D1135V/S1136N/G1218R/R1335Q/T1337R (VNRQR variant);D1135V/G1218R/N1317K/R1335Q/T1337R (VRKQR variant); orD1135V/G1218R/R1335E/T1337R (VRER variant).

In some embodiments, the variant SpCas9 proteins comprise one or moremutations that decrease nuclease activity selected from the groupconsisting of mutations at D10, E762, D839, H983, or D986; and at H840or N863.

In some embodiments, the mutations are: (i) D10A or D10N, and (ii)H840A, H840N, or H840Y.

Also provided herein are isolated Staphylococcus aureus Cas9 (SaCas9)proteins with mutations at one or more of the following positions: E782,N968, and/or R1015, e.g., comprising a sequence that is at least 80%identical to the amino acid sequence of SEQ ID NO:2. Also providedherein are isolated Staphylococcus aureus Cas9 (SaCas9) proteins withmutations at one, two or more of the following positions: E735, E782,K929, N968, A1021, K1044 and/or R1015. In some embodiments, the variantSaCas9 proteins comprise one or more of the following mutations: R1015Q,R1015H, E782K, N968K, E735K, K929R, A1021T, K1044N. In some embodiments,the variant SaCas9 proteins comprise one or more mutations that decreasenuclease activity selected from the group consisting of mutations atD10, D556, H557, and/or N580. In some embodiments, the variant SaCas9proteins comprise mutations at D10A, D556A, H557A, N580A, e.g.,D10A/H557A and/or D10A/D556A/H557A/N580A.

SpCas9 variants described herein can include the amino acid sequence ofSEQ ID NO:1, with mutations at one or more of the following positions:D1135, G1218, R1335, T1337. In some embodiments, the SpCas9 variants caninclude one or more of the following mutations: D1135V; D1135E; G1218R;R1335E; R1335Q; and T1337R. In some embodiments, the SpCas9 variants caninclude one of the following sets of mutations: D1135V/R1335Q/T1337R(VQR variant); D1135V/G1218R/R1335Q.T1337R (VRQR variant);D1135E/R1335Q/T1337R (EQR variant); or D1135V/G1218R/R1335E/T1337R (VRERvariant).

SaCas9 variants described herein can include the amino acid sequence ofSEQ ID NO:2, with mutations at one or more of the following positions:E735, E782, K929, N968, R1015, A1021, and/or K1044. In some embodiments,the SaCas9 variants can include one or more of the following mutations:R1015Q, R1015H, E782K, N968K, E735K, K929R, A1021T, K1044N. In someembodiments, the SaCas9 variants can include one of the following setsof mutations: E782K/N968K/R1015H (KKH variant); E782K/K929R/R1015H (KRHvariant); or E782K/K929R/N968K/R1015H (KRKH variant).

Also provided herein are fusion protein comprising the isolated variantSaCas9 or SpCas9 proteins described herein fused to a heterologousfunctional domain, with an optional intervening linker, wherein thelinker does not interfere with activity of the fusion protein. In someembodiments, the heterologous functional domain is a transcriptionalactivation domain. In some embodiments, the transcriptional activationdomain is from VP64 or NF-κB p65. In some embodiments, the heterologousfunctional domain is a transcriptional silencer or transcriptionalrepression domain. In some embodiments, the transcriptional repressiondomain is a Krueppel-associated box (KRAB) domain, ERF repressor domain(ERD), or mSin3A interaction domain (SID). In some embodiments, thetranscriptional silencer is Heterochromatin Protein 1 (HP1), e.g., HP1αor HP1β. In some embodiments, the heterologous functional domain is anenzyme that modifies the methylation state of DNA. In some embodiments,the enzyme that modifies the methylation state of DNA is a DNAmethyltransferase (DNMT) or a TET protein. In some embodiments, the TETprotein is TET1. In some embodiments, the heterologous functional domainis an enzyme that modifies a histone subunit. In some embodiments, theenzyme that modifies a histone subunit is a histone acetyltransferase(HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), orhistone demethylase. In some embodiments, the heterologous functionaldomain is a biological tether. In some embodiments, the biologicaltether is MS2, Csy4 or lambda N protein. In some embodiments, theheterologous functional domain is FokI.

Also provided herein are isolated nucleic acids encoding the variantSaCas9 or SpCas9 proteins described herein, as well as vectorscomprising the isolated nucleic acids, optionally operably linked to oneor more regulatory domains for expressing the variant SaCas9 or SpCas9proteins described herein. Also provided herein are host cells, e.g.,mammalian host cells, comprising the nucleic acids described herein, andoptionally expressing the variant SaCas9 or SpCas9 proteins describedherein.

Also provided herein are methods of altering the genome of a cell, byexpressing in the cell an isolated variant SaCas9 or SpCas9 proteindescribed herein, and a guide RNA having a region complementary to aselected portion of the genome of the cell.

Also provided herein are methods for altering, e.g., selectivelyaltering, the genome of a cell by expressing in the cell the variantproteins, and a guide RNA having a region complementary to a selectedportion of the genome of the cell.

Also provided are methods for altering, e.g., selectively altering, thegenome of a cell by contacting the cell with a protein variant describedherein, and a guide RNA having a region complementary to a selectedportion of the genome of the cell.

In some embodiments, the isolated protein or fusion protein comprisesone or more of a nuclear localization sequence, cell penetrating peptidesequence, and/or affinity tag.

In some embodiments of the methods described herein, the cell is a stemcell, e.g., an embryonic stem cell, mesenchymal stem cell, or inducedpluripotent stem cell; is in a living animal; or is in an embryo, e.g.,a mammalian, insect, or fish (e.g., zebrafish) embryo or embryonic cell.

Further, provided herein are methods, e.g., in vitro methods, foraltering a double stranded DNA (dsDNA) molecule. The methods includecontacting the dsDNA molecule with one or more of the variant proteinsdescribed herein, and a guide RNA having a region complementary to aselected portion of the dsDNA molecule.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and FIGs, and from the claims.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-J|Evolution and characterization of SpCas9 variants withaltered PAM specificities. A, Rational mutation of the SpCas9 residuesthat make base-specific contacts to the PAM bases is insufficient toalter PAM specificity in the U2OS human cell-based Enhanced GreenFluorescent Protein (EGFP) disruption assay. Disruption frequencies werequantified by flow cytometry; the mean level of disruption observed withthe background control is represented by the dashed red line for thisand subsequent panels (C, G, H, and J); error bars represent s.e.m.,n=3. B, Schematic of the two-plasmid positive selection assay used toalter the PAM specificity of SpCas9. Cleavage of a target site withinthe positive selection plasmid by a functional Cas9/sgRNA complex isnecessary for survival when bacteria are plated on selective media (seealso FIGS. 12A-B). C, Combinatorial assembly and testing of mutationsobtained from the positive selection for SpCas9 variants that can cleavea target site containing an NGA PAM. SpCas9 Variants were paired withsgRNAs that target sites containing either an NGG or an NGA PAM andactivity was assessed using the EGFP disruption assay. Error barsrepresent s.e.m., n=3. D, Schematic of the negative selection assay, inwhich cleavage of the selection plasmid results in cell death whenbacteria are plated on selective media. This system was adapted toprofile the PAM specificity of Cas9 by generating a library of plasmidsthat contain a randomized sequence adjacent to the 3′ end of theprotospacer (see also FIG. 13B). E, Scatterplot of the post-selectionPAM depletion values (PPDVs) of wild-type SpCas9 with two randomized PAMlibraries (each with a different protospacer). PAMs were grouped andplotted by their 2^(nd)/3^(rd)/4^(th) positions. The red dashed lineindicates the cutoff for statistically significant depletion (obtainedfrom a dCas9 control experiment, see FIG. 13C), and the gray dashed linerepresents five-fold depletion (PPDV of 0.2). F, PPDV scatterplots forthe VQR and EQR SpCas9 variants that recognize PAMs distinct from thoserecognized by wild-type SpCas9. G, EGFP disruption frequencies forwild-type, VQR, and EQR SpCas9 on sites with NGAN and NGNG PAMs. Errorbars represent s.e.m., n=3. H, Combinatorial assembly and testing ofmutations obtained from the positive selection for SpCas9 variants thatcan cleave a target site containing an NGCG PAM. sgRNAs that targetsites containing either an NGGG or an NGCG PAM were assessed for Cas9targeting using the EGFP disruption assay. Error bars represent s.e.m.,n=3. I, PPDV scatterplot for the VRER variant. J, EGFP disruptionfrequencies for wild-type and VRER SpCas9 on sites with NGCN and NGNGPAMs. Error bars represent s.e.m., n=3.

FIGS. 2A-F|SpCas9 variants with evolved PAM specificities robustlymodify endogenous sites in zebrafish embryos and human cells. A,Quantification of mutagenesis frequencies in zebrafish embryos inducedby wild-type or VQR SpCas9 on endogenous gene sites bearing NGAG PAMs.Mutation frequencies were determined using the T7E1 assay; error barsrepresent s.e.m., n=5 to 9 individual embryos. B, Mutation frequenciesof the VQR variant quantified by T7E1 assay at 16 target sites in fourendogenous human genes with sgRNAs targeted to sites containing NGAG,NGAT, and NGAA PAMs. Error bars represent s.e.m., n=3. C, Mutationfrequencies of wild-type SpCas9 on endogenous human gene target siteswith NGA PAMs. For ease of comparison, the mutation frequencies for theVQR variant using the same sgRNAs are re-presented here (same data shownin panel B). Error bars represent s.e.m., n=3; n.d., not detectable byT7E1. D, Mutation frequencies of wild-type, VRER, and VQR SpCas9 at ninetarget sites containing NGCG PAMs in three endogenous human genesquantified by T7E1 assay. sgRNA complementarity lengths of 19 and 20 ntwere used; error bars represent s.e.m., n=3. E, Representation of thenumber sites in the human genome with 20 nt spacers targetable bywild-type, VQR, and VRER SpCas9. F, Number of off-target cleavage sitesidentified by GUIDE-seq for the VQR and VRER SpCas9 variants usingsgRNAs from panels B and D.

FIGS. 3A-G|A D1135E mutation improves the PAM recognition and spacerspecificity of SpCas9. A, PPDV scatterplots for wild-type and D1135ESpCas9 (left and right panels, respectively) for the two randomized PAMlibraries. PAMs were grouped and plotted by their 2^(nd)/3^(rd)/4^(th)positions. The data shown for wild-type SpCas9 is the same as the plotfrom FIG. 1D and is re-presented here for ease of comparison. The reddashed line indicates PAMs that are statistically significantly depleted(see FIG. 13C), and the gray dashed line indicates a five-fold depletioncutoff (PPDV of 0.2). B, EGFP disruption activities of wild-type andD1135E SpCas9 on sites that contain NGG, NAG, and NGA PAMs in humancells. Disruption frequencies were quantified by flow cytometry; themean level of disruption observed with the background control isrepresented by the dashed red line for this panel and (D); error barsrepresent s.e.m., n=3; mean fold change in activity is shown. C,Mutagenesis frequencies detected by T7E1 for wild-type and D1135E SpCas9at six endogenous sites in human cells. Error bars represent s.e.m.,n=3; mean fold change in activity is shown. D, Titration of the amountof wild-type or D1135E SpCas9-encoding plasmid transfected for EGFPdisruption experiments in human cells. The amount of sgRNA plasmid usedfor all of these experiments was fixed at 250 ng. Two sgRNAs targetingdifferent EGFP sites were used; error bars represent s.e.m., n=3. E,Targeted deep-sequencing of on- and off-target sites for 3 sgRNAs usingwild-type and D1135E SpCas9 (SEQ ID NOS 660-687, respectively, in orderof appearance). The on-target site is shown at the top, with off-targetsites listed below highlighting mismatches to the on-target. Folddecreases in activity with D1135E relative to wild-type SpCas9 atoff-target sites greater than the change in activity at the on-targetsite are highlighted in green; control indel levels for each ampliconare reported. F, Summary of the targeted deep-sequencing data, plottedas the fold-decrease in activity at on- and off-target sites usingD1135E relative to the indel frequency observed with wild-type SpCas9.G, Summary of GUIDE-seq detected changes in specificity betweenwild-type and D1135E at off-target sites, plotted as the normalizedfold-change in specificity using D1135E versus the read counts at thatoff-target site using wild-type SpCas9 (see also FIG. 18C). Estimatedfold-gain in specificity at sites without read-counts for D1135E are notplotted (see FIG. 18C).

FIGS. 4A-G|Characterization of St1Cas9 and SaCas9 orthologues inbacteria and human cells. A, PPDV scatterplots for St1Cas9 using the tworandomized PAM libraries. PAMs were grouped and plotted by their3^(rd)/4^(th)/5^(th)/6^(th) positions. sgRNA complementarity lengths of20 and 21 nucleotides were used to program St1Cas9 for both libraries(left and right panels, respectively). The red dashed line indicatesPAMs that are statistically significantly depleted (see FIG. 13C), andthe gray dashed line represents five-fold depletion (PPDV of 0.2); α,PAM previously predicted by a bioinformatic approach²⁷; β, PAMspreviously identified under stringent experimental conditions²⁰; *,novel PAMs discovered in this study; γ, PAMs previously identified undermoderate experimental conditions²⁰ (PAM 1 disclosed as SEQ ID NO: 3). B,PPDV scatterplots for SaCas9 using the two randomized PAM libraries.PAMs were grouped and plotted by their 3^(rd)/4^(th)/5^(th)/6^(th)positions. sgRNA complementarity lengths of 21 and 23 nucleotides wereused to program SaCas9 for both libraries (left and right panels,respectively). PAMs identified for SaCas9 are shown, with PAMs 1-3consistently depleted across all combinations of spacer and spacerlength used in these experiments (PAMs 1-3 disclosed as SEQ ID NOS 4, 6and 5, respectively, in order of appearance). C, Survival percentages ofSt1Cas9 and SaCas9 in the bacterial positive selection when challengedwith selection plasmids that harbor different target sites and PAMsindicated on the x-axis. Highly depleted PAMs from panels (A) and (B)for St1Cas9 and SaCas9 were used for the target sites in the positiveselection plasmids (SEQ ID NOS 3, 3, 5, 5, 5 and 3, respectively, inorder of appearance). D, E, EGFP disruption activities of St1Cas9 (panelD) or SaCas9 (panel E) on sites in EGFP that contain NNAGAA (SEQ IDNO:3) or NNGGGT (SEQ ID NO:4)/NNGAGT (SEQ ID NO:5) PAMs, respectively.Matched sgRNAs of different lengths for the same site are indicated;disruption frequencies were quantified by flow cytometry; the meanfrequency of EGFP disruption obtained with a negative control isrepresented by the dashed red line; error bars represent s.e.m., n=3. F,G, Mutation frequencies of St1Cas9 (panel F) and SaCas9 (panel G)quantified by T7E1 assay at sites in four endogenous human genes thatcontain NNAGAA (SEQ ID NO:3) or NNGGGT (SEQ ID NO:4)/NNGAGT (SEQ IDNO:5)/NNGAAT (SEQ ID NO:6) PAMs, respectively. Error bars represents.e.m., n=3; n.d., not detectable by T7E1.

FIGS. 5A-J. Sequences and Maps - plasmids used in this study SEQ ID FIGName NO Description 5A BPK764 7T7-humanSpCas9-NLS-3xFLAG-T7-Bsalcassette-SpgRNA T7 promoters: nts 1-17and 4360-4376; human codon optimized S. pyogenes Cas9 88-4224; NuclearLocalization Signal (NLS) (CCCAAGAAGAAGAGGAAAGTC (SEQ ID NO: 650)) atnts 4198-4218, 3xFLAG tag 4225-4290, Bsal sites 4379-4384 and 4427-4432,gRNA (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 651)) 4434-4509, T7terminator 4252-4572 of SEQ ID NO: 7 MSP712 8T7-humanSpdCas9(D10A/H840A)-T7-Bsalcassette-SpgRNA T7 promoters at nts1-17 and 4360-4376, human codon optimized S. pyogenes Cas9 88-4293,modified codons iat 115-117 and 2605-2607, bold and underlined, NLS(CCCAAGAAGAAGAGGAAAGTC (SEQ ID NO: 650)) at nts 4198-4218, 3xFLAG tag4225-4290, Bsal sites 4379-4384 and 4427-4432, gRNA(GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 651)) at nts4434-4509, T7 terminator 4252-4572 of SEQ ID NO: 8 5B BPK2169 9T7-humanSt1Cas9-NLS-T7-BspMIcassette-St1gRNA T7 promoters at 1-17 and3555-3571, human codon optimized S. thermophilus1 Cas9 at 88-3489, NLSat 3454 to 3486; BspMI sites at 3577-3582 and 3625-3630, gRNA at3635-3763, T7 terminator 3778-3825 of SEQ ID NO: 9. 5C BPK2101 10T7-humanSaCas9-NLS-3xFLAG-T7-BsaIcassette-SagRNA T7 promoters at 1-17and 3418-3434, human codon optimized S. aureus Cas9 at 88-3352, NLS at3256-3276, 3xFLAG tag at 3283-3348, Bsal sites at 3437-3442 and3485-3490, gRNA at 3492-3616, T7 terminator at 3627-2674 of SEQ ID NO:10. 5D p11-lacY — BAD-ccDB-Amp^(R)-AraC-lacY(A177C) wtx1¹⁷ 5E JDS246 11CMV-T7-humanSpCas9-NLS-3xFLAG ADDGENE ID: 43861 Human codon optimized S.pyogenes Cas9 1-4206, NLS at 4111-4131, 3xFLAG tag at 4138-4203 of SEQID NO: 11. MSP469 12 CMV-T7-humanSpCas9(D1135V/R1335Q/T1337R)-NLS-3xFLAG(VQR variant) Human codon optimized S. pyogenes Cas9 1-4206, modifiedcodons at 3403-3405, 4003-4005, and 4009-4011, NLS at 411-4131, 3xFLAGtag 4138-4203 of SEQ ID NO: 12. MSP680 13CMV-T7-humanSpCas9(D1135E/R1335Q/T1337R)-NLS-3xFLAG (EQR variant) Humancodon optimized S. pyogenes Cas9 1-4206, modified codons at 3403-3405,3652-3654, 4003-4005, and 4009-4011, NLS at 411-4131, 3xFLAG tag4138-4203 of SEQ ID NO: 13. MSP1101 14CMV-T7-humanSpCas9(D1135V/G1218R/R1335E/T1337R)-NLS- 3xFLAG (VRERvariant) Human codon optimized S. pyogenes Cas9 1-4206, modified codonsat 3403-3405, 4003-4005, and 4009-4011, NLS at 411-4131, 3xFLAG tag4138-4203 of SEQ ID NO: 14 MSP977 15CMV-T7-humanSpCas9(D1135E)-NLS-3xFLAG Human codon optimized S. pyogenesCas9 1-4206, modified codons at 3403-3405, NLS at 411-4131, 3xFLAG tag4138-4203 of SEQ ID NO: 15. 5F MSP1393 16 CAG-humanSt1Cas9-NLS Humancodon optimized S. thermophilus1 Cas9 1-3402, NLS at 3367-3399 of SEQ IDNO: 16. 5G BPK2139 17 CAG-humanSaCas9-NLS-3xFLAG Human codon optimizedS. aureus Cas9 1-3195, NLS 3169-3189, 3xFLAG tag 3196-3261 of SEQ ID NO:17. 5H BPK1520 18 U6-BsmBIcassette-SpgRNA U6 promoter at 1-318, BsmBIsites at 320-325 and 333-338, S. pyogenes gRNA 339-422, U6 terminator416-422 of SEQ ID NO: 18. 5I BPK2301 19 U6-BsmBIcassette-StlgRNA U6promoter 1-318, BsmBI sites at 320-325 and 333-338, S. thermophilus1gRNA 340-471, U6 terminator 464-471 of SEQ ID NO: 19. 5J VVT1 20U6-BsmBIcassette-SagRNA U6 promoter 1-318, BsmBI sites at 320-325 and333-338, S. aureus gRNA 340-466, U6 terminator 459-466 of SEQ ID NO: 20.

FIG. 6|Alignment of Cas9 orthologues to predict PAM-interacting residuesof SaCas9. The PAM-interacting domains of SpCas9, SaCas9, and 11 otherCas9 orthologues were aligned to identify PAM contacting residues inSaCas9, based on what is known for SpCas9. Top, Top, S. Pyogenes, aminoacids 1229-1368 of SEQ ID NO:1, then SEQ ID NOs:29-40, respectively.

FIG. 7|Substitutions in SaCas9 assessed for activity against differentPAMs in the bacterial screen. Based on the alignment from FIG. 6, singleamino acid substitutions were tested in the bacterial positive selectionto screen for effects on activity against a canonical NNGAGT (SEQ IDNO:5) and non-canonical NNAAGT (SEQ ID NO:41) and NNAGGT (SEQ ID NO:42)PAMs. Bacterial colonies on the selective media suggest that the SaCas9variant has activity against a site containing the indicated PAM.

FIGS. 8A-B|Summary of amino acid substitutions that enable SaCas9variants to target NNARRT (SEQ ID NO:43) PAMs. Amino acid sequences ofthe PAM-interacting domain of 52 selected mutant SaCas9 clones thatenabled survival in bacteria against sites containing an NNARRT (SEQ IDNO:43) PAM; the sequences presented are partial sequences of SEQ IDNOs:53-104 shown in Table 6 (SEQ ID NOS 967-1019, respectively, in orderof appearance). Figure discloses “IIKKG” as SEQ ID NO: 966.

FIG. 9|Human cell activity of wild-type and engineered SaCas9 variants.Activity of wild-type, KKQ, and KKH SaCas9 was assessed in the humancell EGFP reporter assay against sites containing NNRRRT (SEQ ID NO:45)PAMs.

FIG. 10. SaCas9 activity against non-canonical PAMs in bacteria, and howdirected mutations at R1015 impact activity against the samenon-canonical PAMs (SEQ ID NOS 5, 511-514, 5, 511-512, 5, 513-514, 5 and513-514, respectively, in order of appearance).

FIG. 11. Engineered variants can recognize PAMs of the form NNNRRT (SEQID NOS 5, 41-42, and 511-514, respectively, in order of appearance, onboth the left and right side of the figure)

FIGS. 12A-B|Bacterial-based positive selection used to engineer alteredPAM specificity variants of SpCas9. A, Expanded schematic of thepositive selection from FIG. 1B (left panel), and validation that SpCas9behaves as expected in the positive selection (right panel). Spacer 1,SEQ ID NO:105; Spacer 2, SEQ ID NO:106. B, Schematic of how the positiveselection was adapted to select for SpCas9 variants that have alteredPAM recognition specificities. A library of SpCas9 clones withrandomized PAM-interacting (PI) domains (residues 1097-1368) ischallenged by a selection plasmid that harbors an altered PAM (targetsite disclosed as SEQ ID NO: 688). SpCas9 variants that survive theselection by cleaving the positive selection plasmid are sequenced todetermine the mutations that enable altered PAM specificity.

FIGS. 13A-D|Bacterial cell-based site-depletion assay for profiling theglobal PAM specificities of Cas9 nucleases. A, Expanded schematicillustrating the negative selection from FIG. 1D (left panel), andvalidation that wild-type SpCas9 behaves as expected in a screen ofsites with functional (NGG) and non-functional (NGA) PAMs (right panel).B, Schematic of how the negative selection was used as a site-depletionassay to screen for functional PAMs by constructing negative selectionplasmid libraries containing 6 randomized base pairs in place of thePAM. Selection plasmids that contain PAMs cleaved by a Cas9/sgRNA ofinterest are depleted while PAMs that are not cleaved (or poorlycleaved) are retained. The frequencies of the PAMs following selectionare compared to their pre-selection frequencies in the startinglibraries to calculate the post-selection PAM depletion value (PPDV).Spacer 1, SEQ ID NO: 689; Spacer 2, SEQ ID NO: 690. C, D, A cutoff forstatistically significant PPDVs was established by plotting the PPDV ofPAMs for catalytically inactive SpCas9 (dCas9) (grouped and plotted bytheir 2nd/3rd/4th positions) for the two randomized PAM libraries (C). Athreshold of 3.36 standard deviations from the mean PPDV for the twolibraries was calculated (red lines in (D)), establishing that any PPDVdeviation below 0.85 is statistically significant compared to dCas9treatment (red dashed line in (C)). The gray dashed line in (C)indicates a five-fold depletion in the assay (PPDV of 0.2).

FIG. 14|Concordance between the site-depletion assay and EGFP disruptionactivity. Data points represent the average EGFP disruption of the twoNGAN and NGNG PAM sites for the VQR and EQR SpCas9 variants (FIG. 1G)plotted against the mean PPDV observed for library 1 and 2 (FIG. 1F) forthe corresponding PAM. The red dashed line indicates PAMs that arestatistically significantly depleted (PPDV of 0.85, see FIG. 13C), andthe gray dashed line represents five-fold depletion (PPDV of 0.2). Meanvalues are plotted with the 95% confidence interval.

FIG. 15|Insertion or deletion mutations induced by the VQR SpCas9variant at endogenous zebrafish sites containing NGAG PAMs. For eachtarget locus, the wild-type sequence is shown at the top with theprotospacer highlighted in yellow (highlighted in green if present onthe complementary strand) and the PAM is marked as red underlined text.Deletions are shown as red dashes highlighted in gray and insertions aslower case letters highlighted in blue. The net change in length causedby each indel mutation is shown on the right (+, insertion; −,deletion). Note that some alterations have both insertions and deletionsof sequence and in these instances the alterations are enumerated inparentheses. The number of times each mutant allele was recovered (ifmore than once) is shown in brackets.

FIGS. 16A-B|Endogenous genes targeted by wild-type and evolved variantsof SpCas9. A, Sequences targeted by wild-type, VQR, and VRER SpCas9 areshown in blue, red, and green, respectively. Sequences of sgRNAs andprimers used to amplify these loci for T7E1 are provided in Tables 1 and2, below. B, Mean mutagenesis frequencies detected by T7E1 for wild-typeSpCas9 at eight target sites bearing NGG PAMs in the four differentendogenous human genes (corresponding to the annotations in the toppanel). Error bars represent s.e.m., n=3.

FIGS. 17A-B|Specificity profiles of the VQR and VRER SpCas9 variantsdetermined using GUIDE-seq. The intended on-target site is marked with ablack square, and mismatched positions within off-target sites arehighlighted. A, The specificity of the VQR variant was assessed in humancells by targeting endogenous sites containing NGA PAMs: EMX1 site 4(SEQ ID NO:142 and variants disclosed as SEQ ID NOS 691-692,respectively, in order of appearance), FANCF site 1 (SEQ ID NO:143 andvariants disclosed as SEQ ID NOS 693-699, respectively, in order ofappearance), FANCF site 3 (SEQ ID NO:144 and variants disclosed as SEQID NOS 700-702, respectively, in order of appearance), FANCF site 4 (SEQID NO:145 and variant disclosed as SEQ ID NO: 703), RUNX1 site 1 (SEQ IDNO:146 and variants disclosed as SEQ ID NOS 704-714, respectively, inorder of appearance), RUNX1 site 3 (SEQ ID NO:147 and variants disclosedas SEQ ID NOS 715-771, respectively, in order of appearance), VEGFA site1 (SEQ ID NO:148 and variant disclosed as SEQ ID NO: 772), and ZSCAN2(SEQ ID NO:149 and variants disclosed as SEQ ID NOS 773-794,respectively, in order of appearance). B, The specificity of the VRERvariant was assessed in human cells by targeting endogenous sitescontaining NGCG PAMs: FANCF site 3 (SEQ ID NO:150 and variants disclosedas SEQ ID NOS 795-796, respectively, in order of appearance), FANCF site4 (SEQ ID NO:151 and variants disclosed as SEQ ID NOS 797-798,respectively, in order of appearance), RUNX1 site 1 (SEQ ID NO:152 andvariant disclosed as SEQ ID NO: 799), VEGFA site 1 (SEQ ID NO:153 andvariants disclosed as SEQ ID NOS 800-804, respectively, in order ofappearance), and VEGFA site 2 (SEQ ID NO:154 and variant disclosed asSEQ ID NO: 805).

FIGS. 18A-C|Activity differences between D1135E and wild-type SpCas9 atoff-target sites detected by GUIDE-seq. A, Mean frequency of oligo tagintegration at the on-target sites, estimated by restriction fragmentlength polymorphism analysis. Error bars represent s.e.m., n=4. B, Meanmutagenesis frequencies at the on-target sites detected by T7E1. Errorbars represent s.e.m., n=4. C, GUIDE-seq read-count differences betweenwild-type SpCas9 and D1135E at 3 endogenous human cell sites (EMX1 site3 (SEQ ID NO:155 and variants disclosed as SEQ ID NOS 806-812,respectively, in order of appearance); ZNF629 site (SEQ ID NO:156 andvariants disclosed as SEQ ID NOS 813-826, respectively, in order ofappearance), VEGFA site 3 (SEQ ID NO:157 and variants disclosed as SEQID NOS 827-873, respectively, in order of appearance). The on-targetsite is shown at the top and off-target sites are listed below withmismatches highlighted. In the table, a ratio of off-target activity toon-target activity is compared between wild-type and D1135E to calculatethe normalized fold-changes in specificity (with gains in specificityhighlighted in green). For sites without detectable GUIDE-seq reads, avalue of 1 has been assigned to calculate an estimated change inspecificity (indicated in orange). Off-target sites analyzed bydeep-sequencing in FIG. 3E are numbered to the left of the EMX1 site 3and VEGFA site 3 off-target sites.

FIGS. 19A-F|Additional PAMs for St1Cas9 and SaCas9 and activities basedon spacer lengths in human cells. A, PPDV scatterplots for St1Cas9comparing the sgRNA complementarity lengths of 20 and 21 nucleotidesobtained with a randomized PAM library for spacer 1 (top panel) orspacer 2 (bottom panel). PAMs were grouped and plotted by their3rd/4th/5th/6th positions (“nnAGAA” disclosed as SEQ ID NO: 3). The reddashed line indicates PAMs that are statistically significantly depleted(see FIG. 13C) and the gray dashed line represents five-fold depletion(PPDV of 0.2). B, Table of PAMs with PPDVs of less than 0.2 for St1Cas9under each of the four conditions tested. PAM numbering shown on theleft is the same as in FIG. 4A (PAM 1 disclosed as SEQ ID NO: 3). C,PPDV scatterplots for SaCas9 comparing the sgRNA complementarity lengthsof 21 and 23 nucleotides obtained with a randomized PAM library forspacer 1 (top panel) or spacer 2 (bottom panel). PAM were grouped andplotted by their 3rd/4th/5th/6th positions. The red and gray dashedlines are the same as in (A). D, Table of PAMs with PPDVs of less than0.2 for SaCas9 under each of the four conditions tested. PAM numberingis the same as in FIG. 4B (“nnGGGT” disclosed as SEQ ID NO: 4, “nnGAAT”disclosed as SEQ ID NO: 6, “nnGAGT” disclosed as SEQ ID NO: 5, “nnAAGT”disclosed as SEQ ID NO 41, “nnAGGT” disclosed as SEQ ID NO: 42 and“nnCAGT” disclosed as SEQ ID NO: 511). E, F, Human cell activity ofSt1Cas9 and SaCas9 across various spacer lengths via EGFP disruption(panel E, data from FIGS. 4D, 4E) and endogenous gene mutagenesisdetected by T7E1 (panel F, data from FIGS. 4F, 4G). Activity for allreplicates shown (n=3 or 4); bars illustrate mean and 95% confidenceinterval; number of sites per spacer length indicated.

FIGS. 20A-B|Structural and functional roles of D1135, G1218, and T1337in PAM recognition by SpCas9. A, Structural representations of the sixresidues implicated in PAM recognition. The left panel illustrates theproximity of D1135 to S1136, a residue that makes a water-mediated,minor groove contact to the 3rd base position of the PAM15. The rightpanel illustrates the proximity of G1218, E1219, and T1337 to R1335, aresidue that makes a direct, base-specific major groove contact to the3rd base position of the PAM15. Angstrom distances indicated by yellowdashed lines; non-target strand guanine bases dG2 and dG3 of the PAM areshown in blue; other DNA bases shown in orange; water molecules shown inred; images generated using PyMOL from PDB:4UN3. B, Mutational analysisof six residues in SpCas9 that are implicated in PAM recognition. Clonescontaining one of three types of mutations at each position were testedfor EGFP disruption with two sgRNAs targeted to sites harboring NGGPAMs. For each position, we created an alanine substitution and twonon-conservative mutations. S1136 and R1335 were previously reported tomediate contacts to the 3rd guanine of the PAM15, and D1135, G1218,E1219, and T1337 are reported in this study. EGFP disruption activitiesare quantified by flow cytometry; background control represented by thedashed red line; error bars represent s.e.m., n=3.

FIGS. 21A-F Selection and assembly of SaCas9 variants with altered PAMspecificities (A) Phylogenetic tree of Cas9 orthologues with SpCas9 andSaCas9 highlighted. (B) Activity of SaCas9 variants with single aminoacid substitutions assessed in the bacterial positive selection assay(see also FIG. 31B). Error bars represent s.e.m., n=3; NS=no survival.(C) Human cell activity of wild-type and R1015H SaCas9 (SEQ ID NOS 41,511, 5 and 513, respectively, in order of appearance). EGFP disruptionactivity quantified by flow cytometry; error bars represent s.e.m, n=3,mean level of background EGFP loss represented by dashed red line (forthis and panel E). (D) Total number of substitutions observed at eachamino acid position when selecting for SaCas9 variants with altered PAMspecificities (SEQ ID NOS 43 and 47, respectively, in order ofappearance). Starter mutations at R1015 are not counted. (E) Human cellEGFP disruption activity of variants containing mutations observed whenselecting for altered PAM specificities (SEQ ID NOS 41, 5, 42, 4 and511, 513, 512 and 514, respectively, in order of appearance). (F) Meanpost-selection PAM depletion value (PPDV) scatterplot of wild-typeSaCas9 versus the KKH variant (n=2, see also FIG. 34C) (SEQ ID NOS 43,47, 46, 48, 514, 513, 6, 4, 42, 512, 5, 41 and 511, respectively, inorder of appearance). Two libraries with different protospacers and 8randomized basepairs in place of the PAM were used to determine whichPAMs are targetable by each Cas9. Statistically significant depletionindicated by the red dashed line (relative to a dCas9 control, see FIGS.34A and 34B), and 5-fold depletion by the grey dashed line.

FIGS. 22A-F. Activity of the SaCas9 KKH variant targeted to endogenoussites in human cells (A) Mutagenesis frequencies across 55 differentsites bearing NNNRRT PAMs (SEQ ID NOS 43, 47, 46 and 48, respectively,in order of appearance) induced by KKH SaCas9, determined by T7E1 assay.Error bars represent s.e.m., n=3, ND, not detectable by T7E1 assay. (B)KKH variant preference for the third position of the PAM. Meanactivities from data in panel A are shown for this and panels B and C.(C) KKH variant preference for the fourth and fifth positions of thePAM. (D) Spacer length preference of the KKH SaCas9 variant. (E)Comparison of the human cell EGFP disruption activity of wild-type andKKH SaCas9 targeted to various sites containing NNNRRT PAMs (SEQ ID NOS43, 47, 46 and 48, respectively, in order of appearance). EGFPdisruption quantified by flow cytometry; error bars represent s.e.m,n=3, mean level of background EGFP loss represented by dashed red line.(F) Mutagenesis frequencies of wild-type SaCas9 against one site foreach of the 16 possible NNNRRT sites from panel A (sites with thehighest KKH activity were selected). Error bars represent s.e.m., n=3,ND, not detectable by T7E1 assay.

FIGS. 23A-E Genome-wide specificity profiles of wild-type and KKH SaCas9(A) and (B) Direct comparison of wild-type and KKH SaCas9 targeted tosites containing NNGRRT (SEQ ID NO:46) PAMs, represented by total numberof off-targets (panel A) and mismatches observed at each off-target site(panel B) at EMX site 6 (SEQ ID NO:158 and variants disclosed as SEQ IDNOS 874-876, respectively, in order of appearance) and VEGF site 8 (SEQID NO:159 and variants disclosed as SEQ ID NOS 891-925, respectively, inorder of appearance). FANCF site 10 wildtype and variants disclosed asSEQ ID NOS 877 and 878; FANCF site 13 wildtype and variants disclosed asSEQ ID NOS 879-883; RUNX1 site 13 wildtype and variants disclosed as SEQID NOS 884-887; and RUNX1 site 14 variants disclosed as SEQ ID NOS888-890, all respectively, in order of appearance). For panels B and E,GUIDE-seq read counts at each site are indicated; on-target sequencesare marked with a black box; mismatched positions within off-targetsites are highlighted; sequences have been corrected for cell-typespecific SNPs; sites with potential sgRNA or DNA bulge nucleotides areindicated by a small red-bordered base or a dash, respectively. (C) Venndiagram highlighting the differences in off-target site cleavage bywild-type and KKH SaCas9 at VEGFA site 8. (D) and (E) Specificityprofile of the KKH variant targeted to sites containing NNHRRT (SEQ IDNO:44) PAMs, EMX site 1 (SEQ ID NO:160 and variants disclosed as SEQ IDNOS 926-941, respectively, in order of appearance), EMX site 4 (SEQ IDNO:161 and variants disclosed as SEQ ID NOS 942-951, respectively, inorder of appearance), EMX site 10 (SEQ ID NO:162 and variants disclosedas SEQ ID NOS 952-957, respectively, in order of appearance), FANCF site9 (SEQ ID NO:163 and variants disclosed as SEQ ID NOS 958-962,respectively, in order of appearance), and FANCF site 16 (SEQ ID NO:164and variant disclosed as SEQ ID NO: 963), represented by total number ofoff-targets (panel D) and mismatches observed at each off-target site(panel E).

FIG. 24: Activity of VQR-derivative clones in the bacterial 2-plasmidscreen. Testing of 24 different VQR derivative variants against sites inbacteria that contain NGAN PAMs. Survival on the selective plate,relative to the non-selective plate, is indicative of activity againstthe indicated PAM.

FIG. 25: Human cell EGFP disruption activity of SpCas9-VQR derivatives.EGFP disruption activity of the SpCas9 variants is a measure of activityagainst sites that contain the indicated PAM.

FIG. 26: Human cell EGFP disruption activity of SpCas9-VQR and -VRQRvariants. EGFP disruption activity of the SpCas9 variants is a measureof activity against sites that contain the indicated PAM.

FIG. 27: Activity of SpCas9-VRQR derivate variants in the bacterial2-plasmid screen. Testing of 12 different VQR derivative variantsagainst sites in bacteria that contain NGAN PAMs, compared to the VQRand VRQR variants. Survival on the selective plate, relative to thenon-selective plate, is indicative of activity against the indicatedPAM.

FIG. 28: Human cell EGFP disruption activity of SpCas9-VRQR variants.EGFP disruption activity of the SpCas9 variants is a measure of activityagainst sites that contain the indicated PAM.

FIG. 29 Protein domain alignment of Cas9 orthologues (from FIG. 21A).The domain structure of SpCas9 is shown at the top (based on PDB:4UN3;Anders et al., 2014); the PAM contacting residues of SpCas9 arehighlighted; the region of SaCas9 mutagenized to select for altered PAMspecificity variants is shown.

FIGS. 30A-B Primary sequence alignment of Cas9 orthologues foridentification of PAM-interacting residues; SEQ ID NOs:165-176,respectively. SpCas9 residues previously identified (Anders et al.,2014; Examples 1-2) to be important for contacting the PAM arehighlighted in blue, residues capable of modulating SaCas9 PAMspecificity (identified in this study) are highlighted in orange, andpositively charged residues adjacent to R1015 are highlighted in yellow.The structurally predicted PAM-interacting domain of SpCas9 ishighlighted with a blue dashed line (based on PDB:4UN3; Anders et al.,2014), and the conservative estimate of the SaCas9 PAM-interactingdomain used as a boundary for PCR mutagenesis is indicated with anorange dashed line.

FIGS. 31A-B Schematic of the bacterial positive selection assay (A) Theselection plasmids can be modified to screen for Cas9 variants that areable to recognize alternative PAM sequences (SEQ ID NO: 1020). (B)Schematic of the positive selection plasmids (left panel) and expectedoutcomes (right panel) when screening functional or non-functionalCas9/sgRNA pairs in the positive selection (SEQ ID NO: 1021).

FIG. 32 Addition of the K929R mutation to the KNH and KKH variants (SEQID NOS 41-42, 511-512, 5, 4 and 513-514, respectively, in order ofappearance). EGFP disruption activity quantified by flow cytometry;error bars represent s.e.m, n=3, mean level of background EGFP lossrepresented by the dashed red line.

FIG. 33 Schematic of the bacterial site-depletion assay. Site-depletionplasmids with 8 randomized nucleotides in place of the PAM that arerefractory to cleavage by wild-type or KKH SaCas9 are sequenced. Library1 Spacer sequence, SEQ ID NO:964; library 2 spacer sequence, SEQ ID NO:965. Targetable PAMs are inferred by their depletion relative to theinput library, calculated as the post-selection PAM depletion value(PPDV).

FIGS. 34A-E Site-depletion assay results for wild-type and KKH SaCas9(A) PPDV values for dCas9 control experiments on both libraries. The reddashed line indicates statistical significance (PPDV=0.794, see panelB); grey dashed line indicates 5-fold depletion; PPDVs for a windowcomprising the 3^(rd)/4^(th)/5^(th)/6^(th) positions of the PAM areplotted (for this and panel C). (B) Statistically significantpost-selection PAM depletion values (PPDVs) were determined from thedCas9 control experiments in panel A. Statistical significance wasdetermined by setting the threshold at 3.36 times the standarddeviation. (C) Comparison of the PPDVs for wild-type and KKH SaCas9 foreach of the two libraries containing 8 randomized nucleotides in placeof the PAM. Library 1 discloses SEQ ID NOS 514, 513, 6, 4, 42, 512, 5,511 and 41, and Library 2 discloses SEQ ID NOS 513-514, 5-6, 4, 42, 41and 511-512, respectively, in order of appearance. (D) and (E) PAMs andcorresponding PPDV values for all PAMs depleted greater than 5-fold forwild-type and KKH SaCas9, respectively. Sequence motifs are shown forPAMs in two categories: 1) greater than 10 fold or 2) 5- to 10-folddepleted.

FIGS. 35A-D Additional characteristics of endogenous sites targeted byKKH SaCas9 (A) Activity for each of the 55 endogenous site sgRNAs,binned based on the 16 possible NRR motifs of an NNNRRT PAM. Meanactivities from FIG. 2A are shown for this and panels B and C. (B) and(C) Relationship between endogenous gene disruption activity and GCcontent of the spacer and PAM, respectively. (D) Sequence logos for thespacer and PAM of target sites binned based on activity. Sites weregrouped based on mean mutation frequency (from FIG. 2A) into low (0-10%,17 sites), medium (10-30%, 17 sites), or high (>30%, 21 sites) activity.

FIGS. 36A-B On-target tag integration and mutagenesis frequencies forGUIDE-seq experiments (A) Restriction fragment length polymorphism(RFLP) analysis to determine the mean GUIDE-seq tag integrationfrequencies. Error bars represent s.e.m., n=3 (for this and panel B).(B) Mean mutagenesis detected by T7E1 assay.

FIGS. 37A-B A truncated repeat: anti-repeat sgRNA outperforms the fulllength sgRNA, similar to previous results (Ran et al., 2015) (A) Humancell EGFP disruption activity for wild-type SaCas9 against 4 sites thatcontain NNGRRT (SEQ ID NO:46) PAMs (SEQ ID NOS 4, 5 and 4, respectively,in order of appearance). EGFP disruption activity quantified by flowcytometry; error bars represent s.e.m, n=3, mean level of backgroundEGFP loss represented by dashed red line (for this and panel B). (B)Human cell EGFP disruption activity for KKH SaCas9 against 8 sites thatcontain NNNRRT PAMs (SEQ ID NOS 41-42, 511-512, 5, 4 and 513-514,respectively, in order of appearance).

DETAILED DESCRIPTION

Although CRISPR-Cas9 nucleases are widely used for genome editing¹⁻⁴,the range of sequences that Cas9 can cleave is constrained by the needfor a specific protospacer adjacent motif (PAM) in the targetsite^(5, 6). For example, SpCas9, the most robust and widely used Cas9to date, primarily recognizes NGG PAMs. As a result, it can often bedifficult to target double-stranded breaks (DSBs) with the precisionthat is necessary for various genome editing applications. In addition,imperfect PAM recognition by Cas9 can lead to the creation of unwantedoff-target mutations^(7,8). The ability to evolve Cas9 derivatives withpurposefully altered or improved PAM specificities would address theselimitations but, to the present inventors' knowledge, no such Cas9variants have been described.

A potential strategy for improving the targeting range of orthogonalCas9s that recognize extended PAMs is to alter their PAM recognitionspecificities. As described herein, PAM recognition specificity ofSpCas9 can be altered using a combination of structure-guided design anddirected evolution performed with a bacterial cell-based selectionsystem; see Examples 1 and 2. Also described herein are variants thathave been evolved to have relaxed or partially relaxed specificities forcertain positions within the PAM; see Example 3. These variants expandthe utility of Cas9 orthologues that specify longer PAM sequences.

Engineered Cas9 Variants with Altered PAM Specificity

The SpCas9 variants engineered in this study greatly increase the sitesaccessible by wild-type SpCas9, further enhancing the opportunities touse the CRISPR-Cas9 platform to practice efficient HDR, to targetNHEJ-mediated indels to small genetic elements, and to exploit therequirement for a PAM to distinguish between two different alleles inthe same cell. The altered PAM specificity SpCas9 variants canefficiently disrupt endogenous gene sites that are not currentlytargetable by SpCas9 in both zebrafish embryos and human cells,suggesting that they will work in a variety of different cell types andorganisms. Importantly, GUIDE-seq experiments show that the globalprofiles of the VQR and VRER SpCas9 variants are similar to or betterthan those observed with wild-type SpCas9. In addition, the improvedspecificity D1135E variant that we identified and characterized providesa superior alternative to the widely used wild-type SpCas9. D1135E hassimilar activity to wild-type SpCas9 on sites with canonical NGG PAMsbut reduces genome-wide cleavage of off-target sites bearing mismatchedspacer sequences and either canonical or non-canonical PAMs.

All of the SpCas9 and SaCas9 variants described herein can be rapidlyincorporated into existing and widely used vectors, e.g., by simplesite-directed mutagenesis, and because they require only a small numberof mutations contained within the PAM-interacting domain, the variantsshould also work with other previously described improvements to theSpCas9 platform (e.g., truncated sgRNAs (Tsai et al., Nat Biotechnol 33,187-197 (2015); Fu et al., Nat Biotechnol 32, 279-284 (2014)), nickasemutations (Mali et al., Nat Biotechnol 31, 833-838 (2013); Ran et al.,Cell 154, 1380-1389 (2013)), dimeric FokI-dCas9 fusions (Guilinger etal., Nat Biotechnol 32, 577-582 (2014); Tsai et al., Nat Biotechnol 32,569-576 (2014)).

Beyond the mutations to R1335 that presumably contact the 3^(rd) PAMbase position, the SpCas9 variants evolved in this study bear amino acidsubstitutions at D1135, G1218, and T1337, all of which are located nearor adjacent to residues that make direct or indirect contacts to the3^(rd) PAM position in the SpCas9-PAM structure but do not themselvesmediate contacts with the PAM bases (Anders et al., Nature 513, 569-573(2014)) (FIG. 20A). Consistent with this, we found that variousmutations at these positions do not appear to affect SpCas9-mediatedcleavage of sites bearing an NGG PAM (FIG. 20B). These results, togetherwith the nature of the amino acid substitutions at G1218 and T1337 inthe VQR and VRER SpCas9 variants, suggest that alterations at these twopositions may be gain-of-function mutations. For example, it is possiblethat the T1337R mutation is forming backbone or base-specific contactsnear or to the 4^(th) position of the PAM, particularly in the case ofthe VRER variant. The mechanistic role of mutations at D1135 remain lessclear but they may perhaps influence the activity of the adjacent S1136residue, which has been implicated in making a water-mediated contactthrough the minor groove to the guanine in the third position of the PAM(Anders et al., Nature 513, 569-573 (2014)). The D1135E mutation mightimprove specificity by disrupting this network, perhaps reducing theoverall interaction energy of the SpCas9/gRNA complex with the targetsite, a mechanism we have previously proposed might reduce off-targeteffects by making cleavage of these unwanted sequences lessenergetically favorable (Fu et al., Nat Biotechnol 32, 279-284 (2014)).

The present results clearly establish the feasibility of engineeringCas9 nucleases with altered PAM specificities. Characterization ofadditional Cas9 orthologues (Esvelt et al., Nat Methods 10, 1116-1121(2013); Fonfara et al., Nucleic Acids Res 42, 2577-2590 (2014)) orgeneration of domain-swapped Cas9 chimeras (Nishimasu et al., Cell.156(5):935-49 (2014)) as previously described also provide potentialavenues for targeting different PAMs. The engineering strategydelineated herein can also be performed with such orthologues orsynthetic hybrid Cas9s to further diversify the range of targetablePAMs. St1Cas9 and SaCas9 make particularly attractive frameworks forfuture engineering efforts given their smaller sizes relative to SpCas9and our demonstration of their robust genome editing activities in ourbacterial selection systems and in human cells.

Our results strongly suggested that R1015 in wild-type SaCas9 contactsthe G in the third PAM position. Without wishing to be bound by theory,the R1015H substitution may remove this contact and relax specificity atthe third position; however, loss of the R1015 to G contact could alsoconceivably reduce the energy associated with target site binding, whichmay explain why the R1015H mutation alone is not sufficient for robustactivity at NNNRRT sites in human cells. Because the E782K and N968Ksubstitutions both add positive charge, it is possible that they maymake non-specific interactions with the DNA phosphate backbone tocompensate energetically for the loss of the R1015 to guanine contact.

The genetic approach described here does not require structuralinformation and therefore should be applicable to many other Cas9orthologues. The only requirement to evolve Cas9 nucleases withbroadened PAM specificities is that they function in a bacterial-basedselection. While previous studies demonstrated that PAM recognition canbe altered by swapping the PAM-interacting domains of highly relatedCas9 orthologues (Nishimasu et al., Cell (2014)), it remains to bedetermined whether this strategy is generalizable or effective whenusing more divergent orthologues. By contrast, the evolution strategieswe have described herein can be used engineer PAM recognitionspecificities beyond those encoded within naturally occurring Cas9orthologues. This overall strategy can be employed to expand thetargeting range and extend the utility of the numerous Cas9 orthologuesthat exist in nature.

SpCas9 Variants with Altered Specificity

Thus, provided herein are spCas9 variants. The SpCas9 wild type sequenceis as follows:

(SEQ ID NO: 1)        10         20         30         40         50         60MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE        70         80         90        100        110        120ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG       130        140        150        160        170        180NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD       190        200        210        220        230        240VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN       250        260        270        280        290        300LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI       310        320        330        340        350        360LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA       370        380        390        400        410        420GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH       430        440        450        460        470        480AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE       490        500        510        520        530        540VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL       550        560        570        580        590        600SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI       610        620        630        640        650        660IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG       670        680        690        700        710        720RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL       730        740        750        760        770        780HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER       790        800        810        820        830        840MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH       850        860        870        880        890        900IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL       910        920        930        940        950        960TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS       970        980        990       1000       1010       1020KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK      1030       1040       1050       1060       1070       1080MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF      1090       1100       1110       1120       1130       1140ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA      1150       1160       1170       1180       1190       1200YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK      1210       1220       1230       1240       1250       1260YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE      1270       1280       1290       1300       1310       1320QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA      1330       1340       1350       1360 PAAFKYFDTT IDRKRYTSTKEVLDATLIHQ SITGLYETRI DLSQLGGD

The SpCas9 variants described herein can include mutations at one ormore of the following positions: D1135, G1218, R1335, T1337 (or atpositions analogous thereto). In some embodiments, the SpCas9 variantsinclude one or more of the following mutations: D1135V; D1135E; G1218R;R1335E; R1335Q; and T1337R. In some embodiments, the SpCas9 variants areat least 80%, e.g., at least 85%, 90%, or 95% identical to the aminoacid sequence of SEQ ID NO:1, e.g., have differences at up to 5%, 10%,15%, or 20% of the residues of SEQ ID NO:1 replaced, e.g., withconservative mutations. In preferred embodiments, the variant retainsdesired activity of the parent, e.g., the nuclease activity (exceptwhere the parent is a nickase or a dead Cas9), and/or the ability tointeract with a guide RNA and target DNA).

To determine the percent identity of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). The length of a reference sequencealigned for comparison purposes is at least 80% of the length of thereference sequence, and in some embodiments is at least 90% or 100%. Thenucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein nucleic acid “identity” is equivalent to nucleic acid“homology”). The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.Percent identity between two polypeptides or nucleic acid sequences isdetermined in various ways that are within the skill in the art, forinstance, using publicly available computer software such as SmithWaterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol147:195-7); “BestFit” (Smith and Waterman, Advances in AppliedMathematics, 482-489 (1981)) as incorporated into GeneMatcher Plus™,Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure,Dayhof, M. O., Ed, pp 353-358; BLAST program (Basic Local AlignmentSearch Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215:403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2,CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thelength of the sequences being compared. In general, for proteins ornucleic acids, the length of comparison can be any length, up to andincluding full length (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 100%). For purposes of the present compositions andmethods, at least 80% of the full length of the sequence is alignedusing the BLAST algorithm and the default parameters.

For purposes of the present invention, the comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using a Blossum 62 scoring matrix with a gap penalty of 12,a gap extend penalty of 4, and a frameshift gap penalty of 5.

Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine.

In some embodiments, the SpCas9 variants include one of the followingsets of mutations: D1135V/R1335Q/T1337R (VQR variant);D1135V/G1218R/R1335Q/T1337R (VRQR variant); D1135E/R1335Q/T1337R (EQRvariant); or D1135V/G1218R/R1335E/T1337R (VRER variant).

In some embodiments, the SpCas9 variants also include one of thefollowing mutations, which reduce or destroy the nuclease activity ofthe Cas9: D10, E762, D839, H983, or D986 and H840 or N863, e.g.,D10A/D10N and H840A/H840N/H840Y, to render the nuclease portion of theprotein catalytically inactive; substitutions at these positions couldbe alanine (as they are in Nishimasu al., Cell 156, 935-949 (2014)), orother residues, e.g., glutamine, asparagine, tyrosine, serine, oraspartate, e.g., E762Q, H983N, H983Y, D986N, N863D, N863S, or N863H (seeWO 2014/152432). In some embodiments, the variant includes mutations atD10A or H840A (which creates a single-strand nickase), or mutations atD10A and H840A (which abrogates nuclease activity; this mutant is knownas dead Cas9 or dCas9).

Also provided herein are SaCas9 variants. The SaCas9 wild type sequenceis as follows:

(SEQ ID NO: 2)         10         20         30         40 MKRNYILGLDIGITSVGYGI IDYETRDVID AGVRLFKEAN        50         60         70         80 VENNEGRRSK RGARRLKRRRRHRIQRVKKL LFDYNLLTDH         90        100        110        120SELSGINPYE ARVKGLSQKL SEEEFSAALL HLAKRRGVHN       130        140        150        160 VNEVEEDTGN ELSTKEQISRNSKALEEKYV AELQLERLKK        170        180        190        200DGEVRGSINR FKTSDYVKEA KQLLKVQKAY HQLDQSFIDT       210        220        230        240 YIDLLETRRT YYEGPGEGSPFGWKDIKEWY EMLMGHCTYF        250        260        270        280PEELRSVKYA YNADLYNALN DLNNLVITRD ENEKLEYYEK       290        300        310        320 FQIIENVFKQ KKKPTLKQIAKEILVNEEDI KGYRVTSTGK        330        340        350        360PEFTNLKVYH DIKDITARKE IIENAELLDQ IAKILTIYQS       370        380        390        400 SEDIQEELTN LNSELTQEEIEQISNLKGYT GTHNLSLKAI        410        420        430        440NLILDELWHT NDNQIAIFNR LKLVPKKVDL SQQKEIPTTL       450        460        470        480 VDDFILSPVV KRSFIQSIKVINAIIKKYGL PNDIIIELAR        490        500        510        520EKNSKDAQKM INEMQKRNRQ TNERIEEIIR TTGKENAKYL       530        540        550        560 IEKIKLHDMQ EGKCLYSLEAIPLEDLLNNP FNYEVDHIIP        570        580        590        600RSVSFDNSFN NKVLVKQEEN SKKGNRTPFQ YLSSSDSKIS       610        620        630        640 YETFKKHILN LAKGKGRISKTKKEYLLEER DINRFSVQKD        650        660        670        680FINRNLVDTR YATRGLMNLL RSYFRVNNLD VKVKSINGGF       690        700        710        720 TSFLRRKWKF KKERNKGYKHHAEDALIIAN ADFIFKEWKK        730        740        750        760LDKAKKVMEN QMFEEKQAES MPEIETEQEY KEIFITPHQI       770        780        790        800 KHIKDFKDYK YSHRVDKKPNRELINDTLYS TRKDDKGNTL        810        820        830        840IVNNLNGLYD KDNDKLKKLI NKSPEKLLMY HHDPQTYQKL       850        860        870        880 KLIMEQYGDE KNPLYKYYEETGNYLTKYSK KDNGPVIKKI        890        900        910        920KYYGNKLNAH LDITDDYPNS RNKVVKLSLK PYRFDVYLDN       930        940        950        960 GVYKFVTVKN LDVIKKENYYEVNSKCYEEA KKLKKISNQA        970        980        990       1000EFIASFYNND LIKINGELYR VIGVNNDLLN RIEVNMIDIT      1010       1020       1030       1040 YREYLENMND KRPPRIIKTIASKTQSIKKY STDILGNLYE       1050 VKSKKHPQII KKG

The SaCas9 variants described herein include mutations at one or more ofthe following positions: E782, N968, and/or R1015 (or at positionsanalogous thereto). In some embodiments, the variants include one ormore of the following mutations: R1015Q, R1015H, E782K, N968K, E735K,K929R, A1021T, K1044N. In some embodiments, the SaCas9 variants includemutations E782K, K929R, N968K, and R1015X, wherein X is any amino acidother than R. In some embodiments, the SaCas9 variants are at least 80%,e.g., at least 85%, 90%, or 95% identical to the amino acid sequence ofSEQ ID NO:2, e.g., have differences at up to 5%, 10%, 15%, or 20% of theresidues of SEQ ID NO:2 replaced, e.g., with conservative mutations. Inpreferred embodiments, the variant retains desired activity of theparent, e.g., the nuclease activity (except where the parent is anickase or a dead Cas9), and/or the ability to interact with a guide RNAand target DNA).

In some embodiments, the SaCas9 variants also include one of thefollowing mutations, which may reduce or destroy the nuclease activityof the SaCas9: D10A, D556A, H557A, N580A, e.g., D10A/H557A and/orD10A/D556A/H557A/N580A, to render the nuclease portion of the proteincatalytically inactive; substitutions at these positions could bealanine (as they are in Nishimasu al., Cell 156, 935-949 (2014)), orother residues, e.g., glutamine, asparagine, tyrosine, serine, oraspartate. In some embodiments, the variant includes mutations at D10A,D556A, H557A, or N580A (which may create a single-strand nickase), ormutations at D10A/H557A and/or D10A/D556A/H557A/N580A may (which mayabrogate nuclease activity by analogy to SpCas9; these are referred toas dead Cas9 or dCas9).

Also provided herein are isolated nucleic acids encoding the SpCas9and/or SaCas9 variants, vectors comprising the isolated nucleic acids,optionally operably linked to one or more regulatory domains forexpressing the variant proteins, and host cells, e.g., mammalian hostcells, comprising the nucleic acids, and optionally expressing thevariant proteins.

The variants described herein can be used for altering the genome of acell; the methods generally include expressing the variant proteins inthe cells, along with a guide RNA having a region complementary to aselected portion of the genome of the cell. Methods for selectivelyaltering the genome of a cell are known in the art, see, e.g., U.S. Pat.No. 8,697,359; U52010/0076057; US2011/0189776; US2011/0223638;US2013/0130248; WO/2008/108989; WO/2010/054108; WO/2012/164565;WO/2013/098244; WO/2013/176772; US20150050699; US20150045546;US20150031134; US20150024500; US20140377868; US20140357530;US20140349400; US20140335620; US20140335063; US20140315985;US20140310830; US20140310828; US20140309487; US20140304853;US20140298547; US20140295556; US20140294773; US20140287938;US20140273234; US20140273232; US20140273231; US20140273230;US20140271987; US20140256046; US20140248702; US20140242702;US20140242700; US20140242699; US20140242664; US20140234972;US20140227787; US20140212869; US20140201857; US20140199767;US20140189896; US20140186958; US20140186919; US20140186843;US20140179770; US20140179006; US20140170753; Makarova et al., “Evolutionand classification of the CRISPR-Cas systems” 9(6) Nature ReviewsMicrobiology 467-477 (1-23) (June 2011); Wiedenheft et al., “RNA-guidedgenetic silencing systems in bacteria and archaea” 482 Nature 331-338(Feb. 16, 2012); Gasiunas et al., “Cas9-crRNA ribonucleoprotein complexmediates specific DNA cleavage for adaptive immunity in bacteria”109(39) Proceedings of the National Academy of Sciences USA E2579-E2586(Sep. 4, 2012); Jinek et al., “A Programmable Dual-RNA-Guided DNAEndonuclease in Adaptive Bacterial Immunity” 337 Science 816-821 (Aug.17, 2012); Carroll, “A CRISPR Approach to Gene Targeting” 20(9)Molecular Therapy 1658-1660 (September 2012); U.S. Appl. No. 61/652,086,filed May 25, 2012; Al-Attar et al., Clustered Regularly InterspacedShort Palindromic Repeats (CRISPRs): The Hallmark of an IngeniousAntiviral Defense Mechanism in Prokaryotes, Biol Chem. (2011) vol. 392,Issue 4, pp. 277-289; Hale et al., Essential Features and RationalDesign of CRISPR RNAs That Function With the Cas RAMP Module Complex toCleave RNAs, Molecular Cell, (2012) vol. 45, Issue 3, 292-302.

The variant proteins described herein can be used in place of the SpCas9proteins described in the foregoing references with guide RNAs thattarget sequences that have PAM sequences according to the followingTable 4.

TABLE 4 Variant protein Stronger PAM Weaker PAM SpCas9-D1135E NGG NAG,NGA, and NNGG SpCas9-VQR NGAN and NGCG NGGG, NGTG, and NAAG SpCas9-VRQRNGAN SpCas9-EQR NGAG NGAT, NGAA, and NGCG SpCas9-VRER NGCG NGCA, NGCC,and NGCT SaCas9-KKH NNNRRT SaCas9-KKQ NNRRRT (SEQ NNNRRT ID NO: 45)SaCas9-KKE NNCRRT (SEQ NNNRRT ID NO: 47) SaCas9-(KKL NNTRRT (SEQ NNNRRTor KKM) ID NO: 48)

In addition, the variants described herein can be used in fusionproteins in place of the wild-type Cas9 or other Cas9 mutations (such asthe dCas9 or Cas9 nickase described above) as known in the art, e.g., afusion protein with a heterologous functional domains as described in WO2014/124284. For example, the variants, preferably comprising one ormore nuclease-reducing or killing mutation, can be fused on the N or Cterminus of the Cas9 to a transcriptional activation domain or otherheterologous functional domains (e.g., transcriptional repressors (e.g.,KRAB, ERD, SID, and others, e.g., amino acids 473-530 of the ets2repressor factor (ERF) repressor domain (ERD), amino acids 1-97 of theKRAB domain of KOX1, or amino acids 1-36 of the Mad mSIN3 interactiondomain (SID); see Beerli et al., PNAS USA 95:14628-14633 (1998)) orsilencers such as Heterochromatin Protein 1 (HP1, also known as swi6),e.g., HP1α or HP1β; proteins or peptides that could recruit longnon-coding RNAs (lncRNAs) fused to a fixed RNA binding sequence such asthose bound by the MS2 coat protein, endoribonuclease Csy4, or thelambda N protein; enzymes that modify the methylation state of DNA(e.g., DNA methyltransferase (DNMT) or TET proteins); or enzymes thatmodify histone subunits (e.g., histone acetyltransferases (HAT), histonedeacetylases (HDAC), histone methyltransferases (e.g., for methylationof lysine or arginine residues) or histone demethylases (e.g., fordemethylation of lysine or arginine residues)) as are known in the artcan also be used. A number of sequences for such domains are known inthe art, e.g., a domain that catalyzes hydroxylation of methylatedcytosines in DNA. Exemplary proteins include theTen-Eleven-Translocation (TET)1-3 family, enzymes that converts5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in DNA.

Sequences for human TET1-3 are known in the art and are shown in thefollowing table:

GenBank Accession Nos. Gene Amino Acid Nucleic Acid TET1 NP_085128.2NM_030625.2 TET2* NP_001120680.1 (var 1) NM_001127208.2 NP_060098.3 (var2) NM_017628.4 TET3 NP_659430.1 NM_144993.1 *Variant (1) represents thelonger transcript and encodes the longer isoform (a). Variant (2)differs in the 5′ UTR and in the 3′ UTR and coding sequence compared tovariant 1. The resulting isoform (b) is shorter and has a distinctC-terminus compared to isoform a.

In some embodiments, all or part of the full-length sequence of thecatalytic domain can be included, e.g., a catalytic module comprisingthe cysteine-rich extension and the 2OGFeDO domain encoded by 7 highlyconserved exons, e.g., the Tet1 catalytic domain comprising amino acids1580-2052, Tet2 comprising amino acids 1290-1905 and Tet3 comprisingamino acids 966-1678. See, e.g., FIG. 1 of Iyer et al., Cell Cycle. 2009Jun. 1; 8(11):1698-710. Epub 2009 Jun. 27, for an alignment illustratingthe key catalytic residues in all three Tet proteins, and thesupplementary materials thereof for full length sequences (see, e.g.,seq 2c); in some embodiments, the sequence includes amino acids1418-2136 of Tet1 or the corresponding region in Tet2/3.

Other catalytic modules can be from the proteins identified in Iyer etal., 2009.

In some embodiments, the heterologous functional domain is a biologicaltether, and comprises all or part of (e.g., DNA binding domain from) theMS2 coat protein, endoribonuclease Csy4, or the lambda N protein. Theseproteins can be used to recruit RNA molecules containing a specificstem-loop structure to a locale specified by the dCas9 gRNA targetingsequences. For example, a dCas9 variant fused to MS2 coat protein,endoribonuclease Csy4, or lambda N can be used to recruit a longnon-coding RNA (lncRNA) such as XIST or HOTAIR; see, e.g., Keryer-Bibenset al., Biol. Cell 100:125-138 (2008), that is linked to the Csy4, MS2or lambda N binding sequence. Alternatively, the Csy4, MS2 or lambda Nprotein binding sequence can be linked to another protein, e.g., asdescribed in Keryer-Bibens et al., supra, and the protein can betargeted to the dCas9 variant binding site using the methods andcompositions described herein. In some embodiments, the Csy4 iscatalytically inactive. In some embodiments, the Cas9 variant,preferably a dCas9 variant, is fused to FokI as described in WO2014/204578.

In some embodiments, the fusion proteins include a linker between thedCas9 variant and the heterologous functional domains. Linkers that canbe used in these fusion proteins (or between fusion proteins in aconcatenated structure) can include any sequence that does not interferewith the function of the fusion proteins. In preferred embodiments, thelinkers are short, e.g., 2-20 amino acids, and are typically flexible(i.e., comprising amino acids with a high degree of freedom such asglycine, alanine, and serine). In some embodiments, the linker comprisesone or more units consisting of GGGS (SEQ ID NO:188) or GGGGS (SEQ IDNO:189), e.g., two, three, four, or more repeats of the GGGS (SEQ IDNO:188) or GGGGS (SEQ ID NO:189) unit. Other linker sequences can alsobe used.

Expression Systems

To use the Cas9 variants described herein, it may be desirable toexpress them from a nucleic acid that encodes them. This can beperformed in a variety of ways. For example, the nucleic acid encodingthe Cas9 variant can be cloned into an intermediate vector fortransformation into prokaryotic or eukaryotic cells for replicationand/or expression. Intermediate vectors are typically prokaryotevectors, e.g., plasmids, or shuttle vectors, or insect vectors, forstorage or manipulation of the nucleic acid encoding the Cas9 variantfor production of the Cas9 variant. The nucleic acid encoding the Cas9variant can also be cloned into an expression vector, for administrationto a plant cell, animal cell, preferably a mammalian cell or a humancell, fungal cell, bacterial cell, or protozoan cell.

To obtain expression, a sequence encoding a Cas9 variant is typicallysubcloned into an expression vector that contains a promoter to directtranscription. Suitable bacterial and eukaryotic promoters are wellknown in the art and described, e.g., in Sambrook et al., MolecularCloning, A Laboratory Manual (3d ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 2010). Bacterial expressionsystems for expressing the engineered protein are available in, e.g., E.coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene22:229-235). Kits for such expression systems are commerciallyavailable. Eukaryotic expression systems for mammalian cells, yeast, andinsect cells are well known in the art and are also commerciallyavailable.

The promoter used to direct expression of a nucleic acid depends on theparticular application. For example, a strong constitutive promoter istypically used for expression and purification of fusion proteins. Incontrast, when the Cas9 variant is to be administered in vivo for generegulation, either a constitutive or an inducible promoter can be used,depending on the particular use of the Cas9 variant. In addition, apreferred promoter for administration of the Cas9 variant can be a weakpromoter, such as HSV TK or a promoter having similar activity. Thepromoter can also include elements that are responsive totransactivation, e.g., hypoxia response elements, Gal4 responseelements, lac repressor response element, and small molecule controlsystems such as tetracycline-regulated systems and the RU-486 system(see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547;Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, GeneTher., 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahlet al., 1998, Nat. Biotechnol., 16:757-761).

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the nucleic acid inhost cells, either prokaryotic or eukaryotic. A typical expressioncassette thus contains a promoter operably linked, e.g., to the nucleicacid sequence encoding the Cas9 variant, and any signals required, e.g.,for efficient polyadenylation of the transcript, transcriptionaltermination, ribosome binding sites, or translation termination.Additional elements of the cassette may include, e.g., enhancers, andheterologous spliced intronic signals.

The particular expression vector used to transport the geneticinformation into the cell is selected with regard to the intended use ofthe Cas9 variant, e.g., expression in plants, animals, bacteria, fungus,protozoa, etc. Standard bacterial expression vectors include plasmidssuch as pBR322 based plasmids, pSKF, pET23D, and commercially availabletag-fusion expression systems such as GST and LacZ.

Expression vectors containing regulatory elements from eukaryoticviruses are often used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 late promoter, metallothionein promoter, murine mammary tumor viruspromoter, Rous sarcoma virus promoter, polyhedrin promoter, or otherpromoters shown effective for expression in eukaryotic cells.

The vectors for expressing the Cas9 variants can include RNA Pol IIIpromoters to drive expression of the guide RNAs, e.g., the H1, U6 or 7SKpromoters. These human promoters allow for expression of Cas9 variantsin mammalian cells following plasmid transfection.

Some expression systems have markers for selection of stably transfectedcell lines such as thymidine kinase, hygromycin B phosphotransferase,and dihydrofolate reductase. High yield expression systems are alsosuitable, such as using a baculovirus vector in insect cells, with thegRNA encoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of recombinant sequences.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of protein,which are then purified using standard techniques (see, e.g., Colley etal., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification,in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)).Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, 1977, J.Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the known procedures for introducing foreign nucleotide sequencesinto host cells may be used. These include the use of calcium phosphatetransfection, polybrene, protoplast fusion, electroporation,nucleofection, liposomes, microinjection, naked DNA, plasmid vectors,viral vectors, both episomal and integrative, and any of the otherwell-known methods for introducing cloned genomic DNA, cDNA, syntheticDNA or other foreign genetic material into a host cell (see, e.g.,Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingthe Cas9 variant.

The present invention includes the vectors and cells comprising thevectors.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Methods

The following materials and methods were used in Examples 1 and 2.

Plasmids and Oligonucleotides

Schematic maps and DNA sequences for parent constructs used in thisstudy can be found in FIGS. 5A-J and SEQ ID NOs:7-20. Sequences ofoligonucleotides used to generate the positive selection plasmids,negative selection plasmids, and site-depletion libraries are availablein Table 1. Sequences of all gRNA targets in this study are available inTable 2. Point mutations in Cas9 were generated by PCR.

TABLE 1 SEQ ID sequence description NO: Oligos used to generate positiveand negative selection plasmids ctagaGGGCACGGGCAGCTTGCCGGTGGgcatg topoligo to clone site 1 into the 190 positive selection vector (XbaI/SphIcut p11-lacY-wtx1) cCCACCGGCAAGCTGCCCGTGCCCt bottom oligo to clone site1 into the 191 positive selection vectorctagaGGTCGCCCTCGAACTTCACCTCGGgcatg top oligo to clone site 2 into the192 positive selection vector (XbaI/SphI cut p11-lacY-wtx1)cCCGAGGTGAAGTTCGAGGGCGACCt bottom oligo to clone site 2 into the 193positive selection vector aattcGGGCACGGGCAGCTTGCCGGTGGgcatg top oligo toclone site 1 into the 194 negative selection vector (EcoRI/SphI cutp11-lacY-wtx1) cCCACCGGCAAGCTGCCCGTGCCCg bottom oligo to clone site 1into the 195 negative selection vectoraattcGGTCGCCCTCGAACTTCACCTCGGgcatg top oligo to clone site 2 into the196 negative selection vector (EcoRI/SphI cut p11-lacY-wtx1)cCCGAGGTGAAGTTCGAGGGCGACCg bottom oligo to clone site 2 into the 197negative selection vector Oligos used to generate libraries forsite-depletion experiments GCAGgaattcGGGCACGGGCAGCTTGCCGGN top strandoligo for site 1 PAM library, cut 198 NNNNNCTNNNGCGCAGGTCACGAGGCATG withEcoRI once filled in GCAGgaattcGTCGCCCTCGAACTTCACCTN top strand oligofor site 2 PAM library, cut 199 NNNNNCTNNNGCGCAGGTCACGAGGCATG with EcoRIonce filled in /5Phos/CCTCGTGACCTGCGC reverse primer to fill in libraryoligos 200 Primers used to amplify site-depletion libraries forsequencing GATACCGCTCGCCGCAGC forward primer 201CTGCGTTCTGATTTAATCTGTATCAGGC reverse primer 202 Primers used for T7E1experiments GGAGATGTAAATCACCTCCATCTGA forward primer targeted to th1 inzebrafish 203 ATGTTAGCCTACCTCGAAAACCTTC reverse primer targeted to th1in zebrafish 204 CCTGTGCTCTCCTGTTTTTAGGTAT forward primer targeted totia1L in zebrafish 205 AACATGGTAAGAAGCGTGAGTGTTT reverse primer targetedto tia1L in zebrafish 206 CAGGCTGTTGAACCGTAGATTTAGT forward primertargeted to fh in zebrafish 207 TCCACATGTTTTGAGTTTGAGAGTC reverse primertargeted to fh in zebrafish 208 GGAGCAGCTGGTCAGAGGGG forward primertargeted to EMX1 in U2OS human cells 209 CCATAGGGAAGGGGGACACTGG reverseprimer targeted to EMX1 in U2OS human cells 210 GGGCCGGGAAAGAGTTGCTGforward primer targeted to FANCF in U2OS human cells 211GCCCTACATCTGCTCTCCCTCC reverse primer targeted to FANCF in U2OS humancells 212 CCAGCACAACTTACTCGCACTTGAC forward primer targeted to RUNX1 inU2OS human cells 213 CATCACCAACCCACAGCCAAGG reverse primer targeted toRUNX1 in U2OS human cells 214 GATGAGGGCTCCAGATGGCAC forward primertargeted to VEGFA in U2OS human cells 215 GAGGAGGGAGCAGGAAAGTGAGGreverse primer targeted to VEGFA in U2OS human cells 216

TABLE 2 S. pyogenes gRNAs Spacer SEQ length ID SEQ ID Prep Name Name(nt) Sequence NO: Sequence with extended PAM NO: EGFP NXX gRNAs FYF1320NGG 1-20 20 GGGCACGGGCAGCTTGCCGG 217 GGGCACGGGCAGCTTGCCGGTGGT 218BPK1345 NGG 2-20 20 GTCGCCCTCGAACTTCACCT 219 GTCGCCCTCGAACTTCACCTCGGC220 MSP792 NGG 3-20 20 GGTCGCCACCATGGTGAGCA 221 GGTCGCCACCATGGTGAGCAAGGG222 MSP795 NGG 4-20 20 GGTCAGGGTGGTCACGAGGG 223 GGTCAGGGTGGTCACGAGGGTGGG224 FYF1328 NGG 5-20 20 GGTGGTGCAGATGAACTTCA 225GGTGGTGCAGATGAACTTCAGGGT 226 MSP160 NAG 1-20 20 GGGTGGTGCCCATCCTGGTC 227GGGTGGTGCCCATCCTGGTCGAGC 228 MSP161 NAG 2-20 20 GACGTAAACGGCCACAAGTT 229GACGTAAACGGCCACAAGTTCAGC 230 MSP162 NAG 3-20 20 GTGCAGATGAACTTCAGGGT 231GTGCAGATGAACTTCAGGGTCAGC 232 MSP163 NAG 4-20 20 GGGTGGTCACGAGGGTGGGC 233GGGTGGTCACGAGGGTGGGCCAGG 234 MSP164 NAA 1-20 20 GGTCGAGCTGGACGGCGACG 235GGTCGAGCTGGACGGCGACGTAAA 236 MSP165 NAA 2-20 20 GTCGAGCTGGACGGCGACGT 237GTCGAGCTGGACGGCGACGTAAAC 238 MSP168 NGA 1-20 20 GGGGTGGTGCCCATCCTGGT 239GGGGTGGTGCCCATCCTGGTCGAG 240 MSP366 NGA 2-20 20 GCCACCATGGTGAGCAAGGG 241GCCACCATGGTGAGCAAGGGCGAG 242 MSP171 NGA 3-20 20 GTCGCCGTCCAGCTCGACCA 243GTCGCCGTCCAGCTCGACCAGGAT 244 BPK1466 NGA 4-20 20 GCATCGCCCTCGCCCTCGCC245 GCATCGCCCTCGCCCTCGCCGGAC 246 BPK1468 NGA 5-20 20GTTCGAGGGCGACACCCTGG 247 GTTCGAGGGCGACACCCTGGTGAA 248 NGXX gRNAs BPK1468NGAA 1-20 20 GTTCGAGGGCGACACCCTGG 249. GTTCGAGGGCGACACCCTGGTGAA 250.MSP807 NGAA 2-20 20 GTTCACCAGGGTGTCGCCCT 251. GTTCACCAGGGTGTCGCCCTCGAA252. BPK1469 NGAA 3-20 20 GCAGAAGAACGGCATCAAGG 253.GCAGAAGAACGGCATCAAGGTGAA 254. MSP787 NGAA 3-17 17 GAAGAACGGCATCAAGG 255.GAAGAACGGCATCAAGGTGAA 256. MSP170 NGAC 1-20 20 GCCCACCCTCGTGACCACCC 257.GCCCACCCTCGTGACCACCCTGAC 258. MSP790 NGAC 2-20 20 GCCCTTGCTCACCATGGTGG259. GCCCTTGCTCACCATGGTGGCGAC 260. MSP171 NGAT 1-20 20GTCGCCGTCCAGCTCGACCA 261. GTCGCCGTCCAGCTCGACCAGGAT 262. BPK1979 NGAT1-17 17 GCCGTCCAGCTCGACCA 263. GCCGTCCAGCTCGACCAGGAT 264. MSP169 NGAT2-20 20 GTGTCCGGCGAGGGCGAGGG 265. GTGTCCGGCGAGGGCGAGGGCGAT 266. BPK1464NGAT 3-20 20 GGGCAGCTTGCCGGTGGTGC 267. GGGCAGCTTGCCGGTGGTGCAGAT 268.MSP788 NGAT 3-19 19 GGCAGCTTGCCGGTGGTGC 269. GGCAGCTTGCCGGTGGTGCAGAT270. MSP789 NGAT 3-18 18 GCAGCTTGCCGGTGGTGC 271. GCAGCTTGCCGGTGGTGCAGAT272. MSP168 NGAG 1-20 20 GGGGTGGTGCCCATCCTGGT 273.GGGGTGGTGCCCATCCTGGTCGAG 274. MSP783 NGAG 1-19 19 GGGTGGTGCCCATCCTGGT275. GGGTGGTGCCCATCCTGGTCGAG 276. MSP784 NGAG 1-18 18 GGTGGTGCCCATCCTGGT277. GGTGGTGCCCATCCTGGTCGAG 278. MSP785 NGAG 1-17 17 GTGGTGCCCATCCTGGT279. GTGGTGCCCATCCTGGTCGAG 280. MSP366 NGAG 2-20 20 GCCACCATGGTGAGCAAGGG281. GCCACCATGGTGAGCAAGGGCGAG 282. MSP368 NGAG 3-20 20GCCGTAGGTCAGGGTGGTCA 283. GCCGTAGGTCAGGGTGGTCACGAG 284. BPK1974 NGAG3-17 17 GTAGGTCAGGGTGGTCA 285. GTAGGTCAGGGTGGTCACGAG 286. MSP376 NGAG4-20 20 GCTGCCCGACAACCACTACC 287. GCTGCCCGACAACCACTACCTGAG 288. BPK1978NGAG 4-17 17 GCCCGACAACCACTACC 289. GCCCGACAACCACTACCTGAG 290. MSP1028NGCA 1-20 20 GCGAGGGCGATGCCACCTAC 291. GCGAGGGCGATGCCACCTACGGCA 292.MSP1030 NGCA 2-20 20 GTGGTCGGGGTAGCGGCTGA 293. GTGGTCGGGGTAGCGGCTGAAGCA294. MSP1032 NGCC 1-20 20 GGAGCTGTTCACCGGGGTGG 295.GGAGCTGTTCACCGGGGTGGTGCC 296. MSP1033 NGCC 2-20 20 GAACTTGTGGCCGTTTACGT297. GAACTTGTGGCCGTTTACGTCGCC 298. MSP1036 NGCT 1-20 20GGTGAACAGCTCCTCGCCCT 299. GGTGAACAGCTCCTCGCCCTTGCT 300. MSP1037 NGCT2-20 20 GGTGGTGCCCATCCTGGTCG 301. GGTGGTGCCCATCCTGGTCGAGCT 302. MSP800NGCG 1-20 20 GCCACAAGTTCAGCGTGTCC 303. GCCACAAGTTCAGCGTGTCCGGCG 304.MSP801 NGCG 2-20 20 GCGTGTCCGGCGAGGGCGAG 305. GCGTGTCCGGCGAGGGCGAGGGCG306. MSP1360 NGCG 2-18 18 GTGTCCGGCGAGGGCGAG 307. GTGTCCGGCGAGGGCGAGGGCG308. MSP802 NGCG 3-20 20 GCCCGAAGGCTACGTCCAGG 309.GCCCGAAGGCTACGTCCAGGAGCG 310. MSP803 NGCG 4-20 20 GTCGTCCTTGAAGAAGATGG311. GTCGTCCTTGAAGAAGATGGTGCG 312. MSP1366 NGCG 4-17 17GTCCTTGAAGAAGATGG 313. GTCCTTGAAGAAGATGGTGCG 314. MSP792 NGGG 1-20 20GGTCGCCACCATGGTGAGCA 315. GGTCGCCACCATGGTGAGCAAGGG 316. MSP794 NGGG 2-2020 GGTGGTCACGAGGGTGGGCC 317. GGTGGTCACGAGGGTGGGCCAGGG 318. MSP796 NGTG1-20 20 GATCCACCGGTCGCCACCAT 319. GATCCACCGGTCGCCACCATGGTG 320. MSP799NGTG 2-20 20 GTAAACGGCCACAAGTTCAG 321. GTAAACGGCCACAAGTTCAGCGTG 322.Endogenous genes Spacer SEQ length ID SEQ ID Prep Name Name (nt)Sequence NO: Sequence with extended PAM NO: EMX1 FYF1548 NGG 1-20 20GAGTCCGAGCAGAAGAAGAA 323. GAGTCCGAGCAGAAGAAGAAGGGC 324. MSP809 NGG 2-2020 GTCACCTCCAATGACTAGGG 325. GTCACCTCCAATGACTAGGGTGGG 326. MSP811 NGA1-20 20 GAGGAGGAAGGGCCTGAGTC 327. GAGGAGGAAGGGCCTGAGTCCGAG 328. MSP812NGA 2-20 20 GGTTGCCCACCCTAGTCATT 329. GGTTGCCCACCCTAGTCATTGGAG 330.MSP813 NGA 3-20 20 GCTGAGCTGAGAGCCTGATG 331. GCTGAGCTGAGAGCCTGATGGGAA332. MSP814 NGA 4-20 20 GCCACGAAGCAGGCCAATGG 333.GCCACGAAGCAGGCCAATGGGGAG 334. FANCF DR348 NGG 1-20 20GGAATCCCTTCTGCAGCACC 335. GGAATCCCTTCTGCAGCACCTGGA 336. MSP815 NGG 2-2020 GCTGCAGAAGGGATTCCATG 337. GCTGCAGAAGGGATTCCATGAGGT 338. MSP818 NGA1-20 20 GAATCCCTTCTGCAGCACCT 339. GAATCCCTTCTGCAGCACCTGGAT 340. MSP819NGA 2-20 20 GTGCTGCAGAAGGGATTCCA 341. GTGCTGCAGAAGGGATTCCATGAG 342.MSP820 NGA 3-20 20 GCGGCGGCTGCACAACCAGT 343. GCGGCGGCTGCACAACCAGTGGAG344. MSP885 NGA 4-20 20 GGTTGTGCAGCCGCCGCTCC 345.GGTTGTGCAGCCGCCGCTCCAGAG 346. MSP1060 NGCG 1-20 20 GAGGCAAGAGGGCGGCTTTG347. GAGGCAAGAGGGCGGCTTTGGGCG 348. MSP1061 NGCG 2-19 19GGGGTCCAGTTCCGGGATT 349. GGGGTCCAGTTCCGGGATTAGCG 350. MSP1062 NGCG 3-2020 GCAGAAGGGATTCCATGAGG 351. GCAGAAGGGATTCCATGAGGTGCG 352. MSP1063 NGCG4-19 19 GAAGGGATTCCATGAGGTG 353. GAAGGGATTCCATGAGGTGCGCG 354. RUNX1MSP823 NGG 1-20 20 GCTGAAACAGTGACCTGTCT 355. GCTGAAACAGTGACCTGTCTTGGT356. MSP824 NGG 2-20 20 GATGTAGGGCTAGAGGGGTG 357.GATGTAGGGCTAGAGGGGTGAGGC 358. MSP826 NGA 1-20 20 GGTGCATTTTCAGGAGGAAG359. GGTGCATTTTCAGGAGGAAGCGAT 360. MSP827 NGA 2-20 20GTTTTCGCTCCGAAGGTAAA 361. GTTTTCGCTCCGAAGGTAAAAGAA 362. MSP828 NGA 3-2020 GAGATGTAGGGCTAGAGGGG 363. GAGATGTAGGGCTAGAGGGGTGAG 364. MSP829 NGA4-20 20 GCAGAGGGGAGAAGAAAGAG 365. GCAGAGGGGAGAAGAAAGAGAGAT 366. MSP1068NGCG 1-19 19 GGGTGCATTTTCAGGAGGA 367. GGGTGCATTTTCAGGAGGAAGCG 368. VEGFAVC228 NGG 1-20 20 GGTGAGTGAGTGTGTGCGTG 369. GGTGAGTGAGTGTGTGCGTGTGGG370. MSP830 NGG 2-20 20 GTTGGAGCGGGGAGAAGGCC 371.GTTGGAGCGGGGAGAAGGCCAGGG 372. BPK1846 NGA 1-20 20 GCGAGCAGCGTCTTCGAGAG373. GCGAGCAGCGTCTTCGAGAGTGAG 374. BPK1848 NGA 2-20 20GACGTGTGTGTCTGTGTGGG 375. GACGTGTGTGTCTGTGTGGGTGAG 376. BPK1850 NGA 3-2020 GGTTGAGGGCGTTGGAGCGG 377. GGTTGAGGGCGTTGGAGCGGGGAG 378. MSP831 NGA4-20 20 GCTTTGGAAAGGGGGTGGGG 379. GCTTTGGAAAGGGGGTGGGGGGAG 380. MSP1074NGCG 1-20 20 GCAGACGGCAGTCACTAGGG 381. GCAGACGGCAGTCACTAGGGGGCG 382.MSP1075 NGCG 2-20 20 GCTGGGTGAATGGAGCGAGC 383. GCTGGGTGAATGGAGCGAGCAGCG384. MSP1076 NGCG 3-19 19 GTGTGGGTGAGTGAGTGTG 385.GTGTGGGTGAGTGAGTGTGTGCG 386. MSP1077 NGCG 4-19 19 GTGTGCGTGTGGGGTTGAG387. GTGTGCGTGTGGGGTTGAGGGCG 388. S. aureus gRNAs EGFP Spacer SEQ SEQPrep length ID ID Name Name (nt) Sequence NO: Sequence with extended PAMNO: MSP1395 Site 1-20 20 GTCGTGCTGCTTCATGTGGT 389.GTCGTGCTGCTTCATGTGGTCGGGGT 390. MSP1405 Site 1-23 23GAAGTCGTGCTGCTTCATGTGGT 391. GAAGTCGTGCTGCTTCATGTGGTCGGGGT 392. MSP1396Site 2-21 21 GCCGGTGGTGCAGATGAACTT 393. GCCGGTGGTGCAGATGAACTTCAGGGT 394.MSP1397 Site 3-21 21 GCCGTAGGTCAGGGTGGTCAC 395.GCCGTAGGTCAGGGTGGTCACGAGGGT 396. MSP1400 Site 4-21 21GCAACATCCTGGGGCACAAGC 397. GCAACATCCTGGGGCACAAGCTGGAGT 398. MSP1404 Site4-22 22 GGCAACATCCTGGGGCACAAGC 399. GGCAACATCCTGGGGCACAAGCTGGAGT 400.MSP1398 Site 5-21 21 GAAGCACTGCACGCCGTAGGT 401.GAAGCACTGCACGCCGTAGGTCAGGGT 402. MSP1408 Site 5-24 24GCTGAAGCACTGCACGCCGTAGGT 403. GCTGAAGCACTGCACGCCGTAGGTCAGGGT 404.MSP1428 Site 6-21 21 GCCCTCGAACTTCACCTCGGC 405.GCCCTCGAACTTCACCTCGGCGCGGGT 406. MSP1409 Site 6-24 24GTCGCCCTCGAACTTCACCTCGGC 407. GTCGCCCTCGAACTTCACCTCGGCGCGGGT 408.MSP1403 Site 7-22 22 GCAAGGGCGAGGAGCTGTTCAC 409.GCAAGGGCGAGGAGCTGTTCACCGGGGT 410. MSP1406 Site 7-24 24GAGCAAGGGCGAGGAGCTGTTCAC 411. GAGCAAGGGCGAGGAGCTGTTCACCGGGGT 412.MSP1410 Site 8-24 24 GCCCTTCAGCTCGATGCGGTTCAC 413.GCCCTTCAGCTCGATGCGGTTCACCAGGGT 414. S. thermophilus 1 gRNAs EGFP SpacerSEQ SEQ Prep length ID ID Name Name (nt) Sequence NO: Sequence withextended PAM NO: MSP1412 Site 1-20 20 GTCTATATCATGGCCGACAA 415.GTCTATATCATGGCCGACAAGCAGAA 416. MSP1414 Site 2-21 21GCAGCTCGCCGACCACTACCA 417. GCAGCTCGCCGACCACTACCAGCAGAA 418. MSP1417 Site2-23 23 GTGCAGCTCGCCGACCACTACCA 419. GTGCAGCTCGCCGACCACTACCAGCAGAA 420.MSP1413 Site 3-21 21 GCCTTCGGGCATGGCGGACTT 421.GCCTTCGGGCATGGCGGACTTGAAGAA 422. MSP1418 Site 3-24 24GTAGCCTTCGGGCATGGCGGACTT 423. GTAGCCTTCGGGCATGGCGGACTTGAAGAA 424.MSP1416 Site 4-23 23 GTCTATATCATGGCCGACAAGCA 425.GTCTATATCATGGCCGACAAGCAGAAGAA 426. MSP1415 Site 5-23 23GTCTTGTAGTTGCCGTCGTCCTT 427. GTCTTGTAGTTGCCGTCGTCCTTGAAGAA 428. MSP1419Site 5-24 24 GGTCTTGTAGTTGCCGTCGTCCTT 429.GGTCTTGTAGTTGCCGTCGTCCTTGAAGAA 430.

Bacterial Cas9/sgRNA expression plasmids were constructed with two T7promoters to separately express Cas9 and the sgRNA. These plasmidsencode human codon optimized versions of Cas9 for S. pyogenes (BPK764,SpCas9 sequence subcloned from JDS246¹⁷), S. thermophilus Cas9 fromCRISPR locus 1 (MSP1673, St1Cas9 sequence modified from previouspublished description²⁰), and S. aureus (BPK2101, SaCas9 sequence codonoptimized from Uniprot J7RUA5). Previously described sgRNA sequenceswere utilized for SpCas9^(34, 35) and St1Cas9²⁰, while the SaCas9 sgRNAsequence was determined by searching the European Nucleotide Archivesequence HE980450 for crRNA repeats using CRISPRfinder and identifyingthe tracrRNA using a bioinformatic approach similar to one previouslydescribed³⁶. Annealed oligos to complete the spacer complementarityregion of the sgRNA were ligated into BsaI cut BPK764 and BPK2101, orBspMI cut MSP1673 (append 5′-ATAG to the spacer to generate the topoligo and append 5′-AAAC to the reverse compliment of the spacersequence to generate the bottom oligo).

Residues 1097-1368 of SpCas9 were randomly mutagenized using Mutazyme II(Agilent Technologies) at a rate of ˜5.2 substitutions/kilobase togenerate mutagenized PAM-interacting (PI) domain libraries. Thetheoretical complexity of each PI domain library was estimated to begreater than 10⁷ clones based on the number of transformants obtained.Positive and negative selection plasmids were generated by ligatingannealed target site oligos into XbaI/SphI or EcoRI/SphI cutp11-lacy-wtx1¹⁷, respectively.

Two randomized PAM libraries (each with a different protospacersequence) were constructed using Klenow(-exo) to fill-in the bottomstrand of oligos that contained six randomized nucleotides directlyadjacent to the 3′ end of the protospacer (see Table 1). Thedouble-stranded product was cut with EcoRI to leave EcoRI/SphI ends forligation into cut p11-lacY-wtx1. The theoretical complexity of eachrandomized PAM library was estimated to be greater than 10⁶ based on thenumber of transformants obtained.

SpCas9 and SpCas9 variants were expressed in human cells from vectorsderived from JDS246¹⁶. For St1Cas9 and SaCas9, the Cas9 ORFs fromMSP1673 and BPK2101 were subcloned into a CAG promoter vector togenerate MSP1594 and BPK2139, respectively. Plasmids for U6 expressionof sgRNAs (into which desired spacer oligos can be cloned) weregenerated using the sgRNA sequences described above for the SpCas9 sgRNA(BPK1520), the St1Cas9 sgRNA (BPK2301), and the SaCas9 gRNA (VVT1).Annealed oligos to complete the spacer complementarity region of thesgRNA were ligated into the BsmBI overhangs of these vectors (append5′-CACC to the spacer to generate the top oligo and append 5′-AAAC tothe reverse complement of the spacer sequence to generate the bottomoligo).

Bacterial-Based Positive Selection Assay for Evolving SpCas9 Variants

Competent E. coli BW25141(λDE3)²³ containing a positive selectionplasmid (with embedded target site) were transformed withCas9/sgRNA-encoding plasmids. Following a 60 minute recovery in SOBmedia, transformations were plated on LB plates containing eitherchloramphenicol (non-selective) or chloramphenicol+10 mM arabinose(selective). Cleavage of the positive selection plasmid was estimated bycalculating the survival frequency: colonies on selectiveplates/colonies on non-selective plates (see also FIG. 12).

To select for SpCas9 variants that can cleave novel PAMs, PI-domainmutagenized Cas9/sgRNA plasmid libraries were electroporated into E.coli BW25141(λDE3) cells containing a positive selection plasmid thatencodes a target site+PAM of interest. Generally ˜50,000 clones werescreened to obtain between 50-100 survivors. The PI domains of survivingclones were subcloned into fresh backbone plasmid and re-tested in thepositive selection. Clones that had greater than 10% survival in thissecondary screen for activity were sequenced. Mutations observed in thesequenced clones were chosen for further assessment based on theirfrequency in surviving clones, type of substitution, proximity to thePAM bases in the SpCas9/sgRNA crystal structure (PDB:4UN3)¹⁴, and (insome cases) activities in a human cell-based EGFP disruption assay.

Bacterial-Based Site-Depletion Assay for Profiling Cas9 PAMSpecificities

Competent E. coli BW25141(λDE3) containing a Cas9/sgRNA expressionplasmid were transformed with negative selection plasmids harboringcleavable or non-cleavable target sites. Following a 60 minute recoveryin SOB media, transformations were plated on LB plates containingchloramphenicol+carbenicillin. Cleavage of the negative selectionplasmid was estimated by calculating the colony forming units per μg ofDNA transformed (see also FIG. 13).

The negative selection was adapted to determine PAM specificity profilesof Cas9 nucleases by electroporating each randomized PAM library into E.coli BW25141(λDE3) cells that already harbored an appropriate Cas9/sgRNAplasmid. Between 80,000-100,000 colonies were plated at a low densityspread on LB+chloramphenicol+carbenicillin plates. Surviving coloniescontaining negative selection plasmids refractory to cleavage by Cas9were harvested and plasmid DNA isolated by maxi-prep (Qiagen). Theresulting plasmid library was amplified by PCR using Phusion Hot-startFlex DNA Polymerase (New England BioLabs) followed by an AgencourtAmpure XP cleanup step (Beckman Coulter Genomics). Dual-indexed Tru-SeqIllumina deep-sequencing libraries were prepared using the KAPA HTPlibrary preparation kit (KAPA BioSystems) from ˜500 ng of clean PCRproduct for each site-depletion experiment. The Dana-Farber CancerInstitute Molecular Biology Core performed 150-bp paired-end sequencingon an Illumina MiSeq Sequencer.

The raw FASTQ files outputted for each MiSeq run were analyzed with aPython program to determine relative PAM depletion. The program (seeMethods) operates as follows: First, a file dialog is presented to theuser from which all FASTQ read files for a given experiment can beselected. For these files, each FASTQ entry is scanned for the fixedspacer region on both strands. If the spacer region is found, then thesix variable nucleotides flanking the spacer region are captured andadded to a counter. From this set of detected variable regions, thecount and frequency of each window of length 2-6 nt at each possibleposition was tabulated. The site-depletion data for both randomized PAMlibraries was analyzed by calculating the post-selection PAM depletionvalue (PPDV): the post-selection frequency of a PAM in the selectedpopulation divided by the pre-selection library frequency of that PAM.PPDV analyses were performed for each experiment across all possible 2-6length windows in the 6 bp randomized region. The windows we used tovisualize PAM preferences were: the 3 nt window representing the 2^(nd),3^(rd) and 4^(th) PAM positions for wild-type and variant SpCas9experiments, and the 4 nt window representing the 3^(rd), 4^(th),5^(th), 6^(th) PAM positions for St1Cas9 and SaCas9.

Two significance thresholds for the PPDVs were determined based on: 1) astatistical significance threshold based on the distribution of dCas9versus pre-selection library log read count ratios (see FIGS. 13C &13D), and 2) a biological activity threshold based on an empiricalcorrelation between depletion values and activity in human cells. Thestatistical threshold was set at 3.36 standard deviations from the meanPPDV for dCas9 (equivalent to a relative PPDV of 0.85), corresponding toa normal distribution two-sided p-value of 0.05 after adjusting formultiple comparisons (i.e. p=0.05/64). The biological activity thresholdwas set at 5-fold depletion (equivalent to a PPDV of 0.2) because thislevel of depletion serves as a reasonable predictor of activity in humancells (see also FIG. 14). The 95% confidence intervals in FIG. 14 werecalculated by dividing the standard deviation of the mean, by the squareroot of the sample size multiplied by 1.96.

Human Cell Culture and Transfection U2OS.EGFP cells harboring a singleintegrated copy of a constitutively expressed EGFP-PEST reporter gene¹⁵were cultured in Advanced DMEM media (Life Technologies) supplementedwith 10% FBS, 2 mM GlutaMax (Life Technologies),penicillin/streptomycin, and 400 μg/ml of G418 at 37° C. with 5% CO₂.Cells were co-transfected with 750 ng of Cas9 plasmid and 250 ng ofsgRNA plasmid (unless otherwise noted) using the DN-100 program of aLonza 4D-nucleofector according to the manufacturer's protocols. Cas9plasmid transfected together with an empty U6 promoter plasmid was usedas a negative control for all human cell experiments. Target sites forendogenous gene experiments were selected within 200 bp of NGG sitescleavable by wild-type SpCas9 (see FIG. 16A and Table 2).

Zebrafish Care and Injections

Zebrafish care and use was approved by the Massachusetts GeneralHospital Subcommittee on Research Animal Care. Cas9 mRNA was transcribedwith PmeI-digested JDS246 (wild-type SpCas9) or MSP469 (VQR variant)using the mMESSAGE mMACHINE T7 ULTRA Kit (Life Technologies) aspreviously described²¹. All sgRNAs in this study were prepared accordingto the cloning-independent sgRNA generation method²⁴. sgRNAs weretranscribed by the MEGAscript SP6 Transcription Kit (Life Technologies),purified by RNA Clean & Concentrator-5 (Zymo Research), and eluted withRNase-free water.

sgRNA- and Cas9-encoding mRNA were co-injected into one-cell stagezebrafish embryos. Each embryo was injected with ˜2-4.5 nL of solutioncontaining 30 ng/μL gRNA and 300 ng/μL Cas9 mRNA. The next day, injectedembryos were inspected under a stereoscope for normal morphologicaldevelopment, and genomic DNA was extracted from 5 to 9 embryos.

Human Cell EGFP Disruption Assay

EGFP disruption experiments were performed as previously described¹⁶.Transfected cells were analyzed for EGFP expression ˜52 hourspost-transfection using a Fortessa flow cytometer (BD Biosciences).Background EGFP loss was gated at approximately 2.5% for all experiments(graphically represented as a dashed red line).

T7E1 Assay, Targeted Deep-Sequencing, and GUIDE-Seq to QuantifyNuclease-Induced Mutation Rates

T7E1 assays were performed as previously described for human cells¹⁵ andzebrafish²¹. For U2OS.EGFP human cells, genomic DNA was extracted fromtransfected cells ˜72 hours post-transfection using the AgencourtDNAdvance Genomic DNA Isolation Kit (Beckman Coulter Genomics). Targetloci from zebrafish or human cell genomic DNA were amplified using theprimers listed in Table 1. Roughly 200 ng of purified PCR product wasdenatured, annealed, and digested with T7E1 (New England BioLabs).Mutagenesis frequencies were quantified using a Qiaxcel capillaryelectrophoresis instrument (QIagen), as previously described for humancells¹⁵ and zebrafish²¹.

For targeted deep-sequencing, previously characterized on- andoff-target sites (Tsai et al., Nat Biotechnol 33, 187-197 (2015); Fu etal., Nat Biotechnol 31, 822-826 (2013; Fu et al., Nat Biotechnol 32,279-284 (2014)) were amplified using Phusion Hot-start Flex with theprimers listed in Table 1. Genomic loci were amplified for a controlcondition (empty sgRNA), wild-type, and D1135E SpCas9. An AgencourtAmpure XP cleanup step (Beckman Coulter Genomics) was performed prior topooling ˜500 ng of DNA from each condition for library preparation.Dual-indexed Tru-Seq Illumina deep-sequencing libraries were generatedusing the KAPA HTP library preparation kit (KAPA BioSystems). TheDana-Farber Cancer Institute Molecular Biology Core performed 150-bppaired-end sequencing on an Illumina MiSeq Sequencer. Mutation analysisof targeted deep-sequencing data was performed as previously described(Tsai et al., Nat Biotechnol 32, 569-576 (2014)). Briefly, IlluminaMiSeq paired end read data was mapped to human genome reference GRChr37using bwa (Li et al., Bioinformatics 25, 1754-1760 (2009)). High-qualityreads (quality score >=30) were assessed for indel mutations thatoverlapped the target or off-target sites. 1-bp indel mutations wereexcluded from the analysis unless they occurred within 1-bp of thepredicted breakpoint. Changes in activity at on- and off-target sitescomparing D1135E versus wild-type SpCas9 were calculated by comparingthe indel frequencies from both conditions (for rates above backgroundcontrol amplicon indel levels).

GUIDE-seq experiments were performed as previously described (Tsai etal., Nat Biotechnol 33, 187-197 (2015)). Briefly, phosphorylated,phosphorothioate-modified double-stranded oligodeoxynucleotides (dsODNs)were transfected into U2OS cells with Cas9 nuclease along with Cas9 andsgRNA expression plasmids, as described above. dsODN-specificamplification, high-throughput sequencing, and mapping were performed toidentify genomic intervals containing DSB activity. For wild-type versusD1135E experiments, off-target read counts were normalized to theon-target read counts to correct for sequencing depth differencesbetween samples. The normalized ratios for wild-type and D1135E SpCas9were then compared to calculate the fold-change in activity atoff-target sites. To determine whether wild-type and D1135E samples forGUIDE-seq had similar oligo tag integration rates at the intended targetsite, restriction fragment length polymorphism (RFLP) assays wereperformed by amplifying the intended target loci with Phusion Hot-StartFlex from 100 ng of genomic DNA (isolated as described above) usingprimers listed in Table 1. Roughly 150 ng of PCR product was digestedwith 20 U of NdeI (New England BioLabs) for 3 hours at 37° C. prior toclean-up using the Agencourt Ampure XP kit. RFLP results were quantifiedusing a Qiaxcel capillary electrophoresis instrument (QIagen) toapproximate oligo tag integration rates. T7E1 assays were performed fora similar purpose, as described above.

Software - for analyzing PAM depletion MiSeq data Run in the commandprompt (in the directory containing the file) using the command ″pythonPAM_depletion.py″--------------------------------------------------------------------------------------------------------------------------------- import numpy as np import pandas as pd import glob import fnmatchimport os from collections import Counter from Bio.Seq import Seq fromBio import SeqIO import itertools import re from pandas importExcelWriter import Tkinter, tkFileDialog _(——)author_(——) = ″Ved V.Topkar″ _(——)version_(——) = ″1.0″ ″″″ IUPAC_notation_regex describes amapping between certain base characters and the relavent regex string(Useful for parsing out ambiguous base strings) ″″″ IUPAC_notation_regex= { ′N′: ′[ATCG]′, ′Y′: ′[CT]′, ′R′: ′[AG]′, ′W′: ′[AT]′, ′S′: ′[CG]′,′A′: ′A′, ′T′: ′T′, ′C′: ′C′, ′G′: ′G′ } def ambiguous_PAMs(length): ″″″Given an inputted length, return a list of strings describing allpossible PAM sequences NOTE: Returned strings include ambiguous basecharacters ″″″ permutations = itertools.product([′N′, ′A′, ′T′, ′C′,′G′], repeat=length) PAMs = [ ] for item in permutations:PAMs.append(′′.join(item)) return PAMs def unambiguous_PAMs(length):permutations = itertools.product([′A′, ′T′, ′C′, ′G′], repeat=length)PAMs = [ ] for item in permutations: PAMs.append(′′.join(item)) returnPAMs def regex_from_seq(seq): ″″″ Given a sequence with ambiguous basecharacters, returns a regex that matches for the explicit (unambiguous)base characters ″″″ regex = ′′ for c in seq: regex +=IUPAC_notation_regex[c] return regex def regex_match_count(regex,list_of_counts): ″″″ Given a list of strings and a regex, return thenumber of strings in the list that the regex matches. ″″″ c = 0 for itemin list of counts: if re.search(regex, item): c += 1 return c deftabulate_substring_frequencies(pams, indices): ″″″ Given a list of rawpams and substring indices, tabulates the frequency oftabulate_substring_frequencies RETURNS a Pandas Series ″″″ base_PAMs =unambiguous_PAMs(indices[1] − indices[0]) tmp_PAMs =Counter([pam[indices[0]:indices[1]] for pam in pams]) c = Counter( ) forbase_PAM in base_PAMs: c[base_PAM] = tmp_PAMs[base_PAM] PAMs =pd.Series(c) PAMs.sort(ascending=False) excel_PAMs = pd.DataFrame( )excel_PAMs[′PAM′] = PAMs.index excel_PAMs[′Count′] = PAMs.valuesexcel_PAMs[′Frequency′] = PAMs.values.astype(float)/sum(PAMs.values)return excel_PAMs def generate_raw_PAM_counts(filepaths, targetsites,PAM_length): ″″″ Here, we get all of our relavent PAM sequences from theinputted files by searching for the targetsites and looking at theflanking region ″″″ reverse_target_sequences = {targetsite:str(Seq(targetsites[targetsite]).reverse_complement( )) for targetsitein targetsites} all_pams = {targetsite: [ ] for targetsite intargetsites} # Iterate through each file and collect the PAMs of eachsequence # Checks both forward and reverse reads for filepath infilepaths: print ′Scanning file: ′ + os.path.basename(filepath) pams = [] records = SeqIO.parse(filepath, filepath. split(′.′)[−1]) for recordin records: seq = str(record.seq) for targetsite in targetsites:target_seq = targetsites[targetsite] target =seq.find(targetsites[targetsite]) if target > −1: index = target +len(target_seq) all_pams[targetsite].append(seq[index:index +PAM_length]) else: target =seq.find(reverse_target_sequences[targetsite]) if target > −1: index =target all_pams[targetsite].append(str(Seq(seq[index −PAM_length:index]).reverse_complement( ))) return all_pams def analyzePAM_depletion_data(filepaths, targetsites, PAM_length=3): ″″″ Given adirectory that contains a given file extension and a target sequence, dothe entire PAM depletion analysis ″″″ # Make sure that dirnames andtarget sequences are inputted if filepaths is None: raiseException(′Please specify a directory name′) if targetsites is None:raise Exception(′Please specify a target sequence′) if PAM_length isNone or PAM_length < 3: raise Exception(′Please enter a valid PAMlength′) all_pams = generate_raw_PAM_counts(filepaths, targetsites,PAM_length) letters = [′A′, ′T′, ′C′ , ′G′] all counters = {targetsite:Counter(all_pams[targetsite]) for targetsite in targetsites} fortargetsite in targetsites: pams = all_pams[targetsite] base_counters =[Counter( ) for x in range(PAM_length)] for pam in pams: for i, c inenumerate(pam): base_counters[i][c] += 1 raw_PAM_counts =pd.Series(all_counters[targetsite]) raw_PAM_counts.sort(ascending=False)raw_counts_df = pd.DataFrame( ) raw_counts_df[′PAM′] =raw_PAM_counts.index raw_counts_df[′Count′] = raw_PAM_counts.valuessingle_base_counts = pd.DataFrame(base_counters) single_base_frequencies= single_base_counts.divide(single_base_counts.sum(axis=1).ix[0]) #Prepare substring counts and frequencies writer = ExcelWriter(′out/′ +os.path.basename(filepath).split(′.′)[0] + ′_′ + targetsite + ′.xlsx′)single_base_counts.to_excel(writer, ′Single Base Counts′)single_base_frequencies.to excel(writer, ′Single Base Frequencies′)raw_counts_df.to_excel(writer, ′Raw PAM Counts′) # Designate whichwindows should be analyzed and name them settings = { ′XXXNNN′: [0,3],′NXXXNN′: [1,4], ′NNXXXN′: [2,5], ′NNNXXX′: [3,6], ′XXXXNN′: [0,4],′NXXXXN′: [1,5], ′NNXXXX′: [2,6], ′XXNNNN′: [0,2], ′NXXNNN′: [1,3],′NNXXNN′: [2,4], ′NNNXXN′: [3,5], ′NNNNXX′: [4,6], ′XXXXXN′: [0,5],′NXXXXX′: [1,6], ′XXXXXX′: [0,6],  } for item in settings: df = tabulatesubstring frequencies(pams, settings[item]) df.to_excel(writer, item)writer.save( ) print ′Saved excel output for ′ + targetsite if_(——)name_(——) == ″_(——)main_(——)″: # Display the filepicker, acceptingonly FASTQ files root = Tkinter.Tk( ) root.withdraw( ) file_paths =tkFileDialog.askopenfilenames(parent=root, title=′Choose FASTQ Files′,filetypes=[(″FastQ files″, ″*.fastq″)]) # Describe the targetsite(s) tosearch for targetsites = {′EGFP site 1′: ′GTCGCCCTCGAACTTCACCT′} # Runthe analysis on the inputted filepaths and targetsite for a givenvariable nucleotide region length analyze_PAM_depletion_data(file_paths,targetsites, PAM_length=6)------------------------------------------------------------------------------------------------------

Example 1

One potential solution to address targeting range limitations would beto engineer Cas9 variants with novel PAM specificities. A previousattempt to alter PAM specificity utilized structural information aboutbase-specific SpCas9-PAM interactions to mutate arginine residues (R1333and R1335) that contact guanine nucleotides at the second and third PAMpositions, respectively (Anders et al., Nature 513, 569-573 (2014)).Substitution of both arginines with glutamines (whose side-chains mightbe expected to interact with adenines) failed to yield SpCas9 variantsthat could cleave targets harboring the expected NAA PAM in vitro(Anders et al., Nature 513, 569-573 (2014)). Using a human cell-basedU2OS EGFP reporter gene disruption assay in which nuclease-inducedindels lead to loss of fluorescence (Reyon et al., Nat Biotechnol 30,460-465 (2012); Fu et al., Nat Biotechnol 31, 822-826 (2013)), weconfirmed that an R1333Q/R1335Q SpCas9 variant failed to efficientlycleave target sites with NAA PAMs (FIG. 1A). Additionally, we found thatsingle R1333Q and R1335Q SpCas9 variants each failed to efficientlycleave target sites with their expected NAG and NGA sites, respectively(FIG. 1A). We therefore reasoned that re-engineering PAM specificitymight require additional mutations at positions other than R1333 andR1335. For example, available structural information shows that K1107and S1136 make direct and indirect minor groove contacts to the secondand third bases in the PAM, respectively (Anders et al., Nature 513,569-573 (2014)). Therefore, it is plausible that additional alterationsat or near these positions might be needed to alter PAM specificity.

To identify additional positions that might be critical for modifyingPAM specificity, we adapted a bacterial selection system previously usedto study properties of homing endonucleases (hereafter referred to asthe positive selection) (Chen & Zhao, Nucleic Acids Res 33, e154 (2005);Doyon et al., J Am Chem Soc 128, 2477-2484 (2006)). In our adaptation ofthis system, Cas9-mediated cleavage of a positive selection plasmidencoding an inducible toxic gene enables cell survival, due tosubsequent degradation and loss of the linearized plasmid (FIG. 1B andFIG. 12A). After establishing that SpCas9 can function in the positiveselection system, we tested both wild-type and the R1335Q variant fortheir ability to cleave a selection plasmid harboring a target site withan NGA PAM and failed to observe survival, as expected (FIG. 12A). Toscreen for gain-of-function mutations, we generated libraries ofwild-type and R1335Q SpCas9 bearing randomly mutagenized PAM-interactingdomains (amino acid positions 1097-1368) with a mean rate of 5.2mutations per kilobase (FIG. 12B and Methods). These libraries wereintroduced into bacteria with a positive selection plasmid containing atarget site with an NGA PAM and plated on selective medium. Sequences ofsurviving clones from the R1335Q-based library revealed that the mostfrequent substitutions in addition to the pre-existing R1335Q mutationwere D1135V/Y/N/E and T1337R (Table 3). We obtained fewer survivors withthe wild-type SpCas9-based library selection but the sequences of theseclones also included D1135V/Y/N and R1335Q mutations. We next assembledand tested SpCas9s bearing all possible single, double, and triplecombinations of the D1135V/Y/N/E, R1335Q, and T1337R mutations using thehuman cell-based EGFP disruption assay. This analysis showed that SpCas9variants with substitutions at all three positions displayed the highestactivities on an NGA PAM, but also the lowest activities on an NGG PAM(FIG. 1C). We chose two SpCas9 variants, D1135V/R1335Q/T1337R andD1135E/R1335Q/T1337R (hereafter referred to as the VQR and EQR SpCas9variants, respectively), because they possessed the greatestdiscrimination between NGA and NGG PAMs (FIG. 1C), for furthercharacterization.

To assess the global PAM specificity profiles of our novel SpCas9variants, we used a bacterial-based negative selection system (FIG. 1Dand FIG. 13A). Previous studies have used similar types of selectionsystems to identify the cleavage site preferences of Cas9 nucleases(Jiang et al., Nat Biotechnol 31, 233-239 (2013); Esvelt et al., NatMethods 10, 1116-1121 (2013)). In our version of this assay (which werefer to as the site-depletion assay), a library of plasmids bearingrandomized 6 bp sequences placed adjacent to a protospacer is tested forcleavage by a Cas9/sgRNA complex in E. coli (FIG. 13B). Plasmids withprotospacer-adjacent sequences resistant to cleavage by a Cas9/sgRNAcomplex enable cell survival due to the presence of an antibioticresistance gene, whereas plasmids bearing cleavable sequences aredegraded and therefore depleted from the library (FIG. 13B).High-throughput sequencing of 100,000 non-targetable sequences enabledus to calculate a post-selection PAM depletion value (PPDV) for anygiven PAM. The PPDV of a PAM (or group of PAMs) is defined as thefrequency of that PAM in the post-selection population divided by itsfrequency in the pre-selection library. This quantitative value providesan estimate of Cas9 activity on that PAM. Profiles obtained withcatalytically inactive Cas9 (dCas9) on two randomized PAM libraries(each with a different protospacer) enabled us to define what representsa statistically significant change in PPDV for any given PAM or group ofPAMs (FIG. 13C). We then validated our site-depletion assay bydemonstrating that the PPDVs for wild-type SpCas9 obtained with the tworandomized PAM libraries recapitulated its previously described profileof targetable PAMs (Jiang et al., Nat Biotechnol 31, 233-239 (2013))(FIG. 1E).

Using the site-depletion assay, we obtained PAM specificity profiles forthe VQR and EQR SpCas9 variants using the two randomized PAM libraries.The VQR variant strongly depleted sites bearing NGAN and NGCG PAMs, andmore weakly NGGG, NGTG, and NAAG PAMs (FIG. 1F). In contrast, the EQRvariant strongly depleted NGAG PAMs and more weakly NGAT, NGAA, and NGCGPAMs (FIG. 1F), demonstrating a potentially more limited targeting rangerelative to the VQR variant. To test whether PAMs identified by thesite-depletion assay could also be recognized in human cells, weassessed cleavage by the VQR and EQR SpCas9 variants on target sitesusing the EGFP disruption assay. The VQR variant robustly cleaved sitesin EGFP bearing NGAN PAMs (with relative efficienciesNGAG>NGAT=NGAA>NGAC), and also sites bearing NGCG, NGGG, and NGTG PAMswith generally lower efficiencies (FIG. 1G). The EQR variant alsorecapitulated its preference for NGAG and NGNG PAMs over the other NGANPAMs in human cells, again all at lower activities than with the VQRvariant (FIG. 1G). Collectively, these results in human cells stronglymirror what was observed with the bacterial site-depletion assay (FIG.14) and suggested that PPDVs of 0.2 (representing a five-fold depletion)in the bacterial assay provide a reasonable predictive threshold foractivity in human cells (FIG. 14).

We next sought to extend the generalizability of our engineeringstrategy by attempting to identify SpCas9 variants capable ofrecognizing an NGC PAM. We first designed Cas9 mutants bearing aminoacid substitutions of R1335 that might be expected to interact with acytosine (D, E, S, or T) and found no activity on an NGC PAM site usingthe positive selection system. We then randomly mutagenized thePAM-interacting domain of each of these singly substituted SpCas9variants but still failed to obtain surviving colonies in positiveselections. Because the T1337R mutation had increased the activities ofour VQR and EQR SpCas9 variants (FIG. 1C), we combined this mutationwith R1335 substitutions of A, D, E, S, T, or V, and again randomlymutagenized their PAM-interacting domains. Selections using two of thesesix mutagenized libraries (bearing pre-existing R1335E/T1337R andR1335T/T1337R substitutions) yielded surviving colonies harboring avariety of additional mutations (Table 3). Characterization of variousselected clones using both bacterial and human cell-based assayssuggested that substitutions at four positions in particular (D1135V,G1218R, R1335E, and T1337R) appeared to be important for cleavage of NGCPAMs. Assembly and testing of all potential single, double, triple, andquadruple combinations of these mutations using the EGFP disruptionassay established that the quadruple VRER variant displayed the highestactivity on an NGCG PAM and minimal activity on an NGGG PAM (FIG. 1H).Analysis of the VRER variant using the site-depletion assay revealed itto be highly specific for NGCG PAMs (FIG. 1I). Consistent with thisresult, EGFP disruption assays performed in human cells with the VRERvariant revealed efficient cleavage of sites with NGCG PAMs, greatlydecreased and inconsistent cleavage of sites with NGCA, NGCC, and NGCTPAMs, and essentially no activity on sites with NGAG, NGTG, and NGGGPAMs (FIG. 1J).

To demonstrate directly that our VQR and VRER SpCas9 variants can enabletargeting of sites not currently modifiable by wild-type SpCas9, wetested their activities on endogenous genes in zebrafish embryos andhuman cells. In single cell zebrafish embryos, we found that the VQRvariant could efficiently modify endogenous gene sites bearing NGAG PAMswith mean mutagenesis frequencies of 20 to 43% (FIG. 2A) and that theindels originated at the predicted cleavage sites (FIG. 15). In humancells, we found that the VQR variant robustly modified 16 sites acrossfour different endogenous genes that harbored NGAG, NGAT, and NGAA PAMs(range of 6 to 53%, mean of 33%; FIG. 2B and FIG. 16A). Importantly, weverified that wild-type SpCas9 was unable to efficiently alter most ofthe same sites with NGAG and NGAT PAMs in zebrafish and human cells(FIGS. 2A and 2C), yet was able to efficiently modify nearby sitesbearing NGG PAMs (FIG. 16B). Similarly, when examining VRER variantactivity at nine sites with NGCG PAMs across three endogenous humangenes, we also observed robust mean disruption frequencies (range of 5to 36%, mean of 21%; FIG. 2D). Consistent with our site-depletion data(FIGS. 1E & 1F), the VQR variant altered NGCG PAM sites efficienciessimilar to that observed with the VRER variant, while wild-type SpCas9was unable to do so (FIG. 2D). Computational analysis of the referencehuman genome sequence shows that the addition of our VQR and VRER SpCas9variants doubles the range of potential target sites compared with whatwas previously possible with only wild-type SpCas9 (FIG. 2E). Takentogether, these results demonstrate that our engineered SpCas9 variantsexpand the targeting range of SpCas9 by enabling modification ofpreviously inaccessible endogenous sites in zebrafish embryos and humancells.

To determine the genomewide specificity of our VQR and VRER SpCas9nucleases, we used the recently described GUIDE-seq (Genome-wideUnbiased Identification of Double-stranded breaks Enabled by sequencing)method¹⁰ to profile off-target cleavage events of these SpCas9 variantsin human cells. We profiled the genome-wide activities of the VQR andVRER SpCas9 variants using a total of 13 different sgRNAs (eight for VQRand five for VRER from FIGS. 2B and 2D, respectively), which we hadshown could induce high efficiencies of modification at their intendedon-target sites. These GUIDE-seq experiments yielded a number ofimportant observations: The numbers of off-target DSBs induced by ourSpCas9 variants in human cells are comparable to (or, in the case of theVRER variant, perhaps even better than) what has been previouslyobserved with wild-type SpCas9 (FIG. 2F). We note that the highgenome-wide specificities observed with VRER might result both from itsrestricted specificity for NGCG PAMs and perhaps from the relativedepletion of sites with NGCG PAMs in the human genome (FIG. 2E)²¹.Additionally, the off-target sites observed generally possess theexpected PAM sequences predicted by our site-depletion experiments,including some tolerance for PAMs “shifted” 3′ by one base (compare PAMsfrom FIGS. 1F and 1I with those in the sites of FIG. 17). Finally, theposition and numbers of mismatches found in the off-target sites of ourVQR and VRER SpCas9 variants (FIG. 17) are similar in theirdistributions to what we previously observed with wild-type SpCas9 forsgRNAs targeted to non-repetitive sequences¹⁰.

Previous studies have shown that imperfect PAM recognition by SpCas9 canlead to recognition of unwanted sites that contain non-canonical NAG,NGA, and other PAMs in human cells (Hsu et al., Nat Biotechnol 31,827-832 (2013); Tsai et al., Nat Biotechnol 33, 187-197 (2015); Jiang etal., Nat Biotechnol 31, 233-239 (2013); Mali et al., Nat Biotechnol 31,833-838 (2013); Zhang et al., Sci Rep 4, 5405 (2014)). Therefore, wewere interested in exploring if mutations at or near residues thatmediate PAM-interaction might improve SpCas9 PAM specificity. Whileengineering the VQR variant we had noticed that a D1135E SpCas9 mutantappeared to better discriminate between a canonical NGG PAM and anon-canonical NGA PAM compared to wild-type SpCas9 (FIG. 1C). Given thisobservation, we comprehensively assessed the PAM recognition profile ofthis D1135E variant using our site-depletion assay. This experimentrevealed a decrease in depletion of non-canonical NAG, NGA, and NNGGPAMs with D1135E SpCas9 relative to wild-type SpCas9 (FIG. 3A).Interestingly, this effect was more prominent for one of the twoprotospacers we used, suggesting that the impact of the D1135Esubstitution on non-canonical PAM recognition may vary to some degree ina protospacer-dependent manner. Importantly, we did not observe theemergence of any new non-canonical PAM specificities.

We next tested whether the improved PAM specificity of D1135E SpCas9also could be observed in human cells. In direct comparisons ofwild-type and D1135E SpCas9 on eight target sites with non-canonical NAGor NGA PAMs, we observed that these sites were consistently lessefficiently cleaved by D1135E than by wild-type SpCas9 in the EGFPdisruption assay (FIG. 3B, mean fold-decrease in activity of 1.94).Importantly, wild-type and D1135E SpCas9 both showed comparableactivities on four EGFP reporter gene sites and six endogenous humangene sites with canonical NGG PAMs (FIGS. 3B and 3C, respectively),demonstrating that the D1135E variant does not appreciably affectcleavage of on-target sites with NGG PAMs (mean fold-decrease inactivity of 1.04 across all ten sites). Titration experiments in whichwe decreased the concentration of Cas9-encoding plasmid transfected intohuman cells revealed no substantial differences in the activities ofwild-type and D1135E SpCas9 when they were targeted to the same sites(FIG. 3D), implying that the increased specificity observed with theD1135E variant is not simply the result of protein destabilization.

To more directly assess whether the introduction of D1135E could reduceoff-target cleavage effects of SpCas9, we used deep-sequencing tocompare mutation rates induced by wild-type and D1135E SpCas9 on 25previously known off-target sites of three different sgRNAs (Hsu et al.,Nat Biotechnol 31, 827-832 (2013); Tsai et al., Nat Biotechnol 33,187-197 (2015); Fu et al., Nat Biotechnol 31, 822-826 (2013)). These 25sites included off-target sites with various mismatches in the spacersequence and both canonical NGG and non-canonical PAMs (FIG. 3E). Theresults of these deep-sequencing experiments revealed that the D1135Evariant showed reduced mutation frequencies at 19 of the 22 off-targetsites with activity above background indel rates, relative to themutation frequency observed at the three on-target sites (FIGS. 3E &3F). Interestingly, these reduced off-target mutation frequencies wereobserved at many sites with a canonical PAM, suggesting that the gain inspecificity with D1135E is not restricted only to sites withnon-canonical PAMs. To assess the improvements in specificity associatedwith D1135E on a genome-wide scale, we performed GUIDE-seq experimentsusing wild-type and D1135E SpCas9 with three different sgRNAs (two ofwhich were previously known to have off-target sites with canonical andnon-canonical PAMs (Hsu et al., Nat Biotechnol 31, 827-832 (2013); Tsaiet al., Nat Biotechnol 33, 187-197 (2015); Fu et al., Nat Biotechnol 31,822-826 (2013)). We observed a generalized improvement in genome-widespecificity when using the D1135E SpCas9 variant compared with wild-typeSpCas9 (FIG. 3G). For all three sgRNAs we tested, these improvements inspecificity were observed at off-target sites that contained mismatchedspacers with canonical or non-canonical PAMs (FIG. 18). Importantly,these GUIDE-seq experiments demonstrated that the introduction of theD1135E mutation does not increase the number of off-target effectsinduced by SpCas9. Collectively, these results show that the D1135Esubstitution can increase the global specificity of SpCas9.

Although all of the experiments described above were performed withSpCas9, there are many Cas9 orthologues from other bacteria that couldmake attractive candidates for characterizing and engineering Cas9s withnovel PAM specificities (Fonfara et al., Nucleic Acids Res 42, 2577-2590(2014); Ran et al., Nature 520, 186-191 (2015)). To explore thefeasibility of doing this, we determined whether two smaller-sizeorthologues, Streptococcus thermophilus Cas9 from the CRISPR1 locus(St1Cas9) (Deveau et al., J Bacteriol 190, 1390-1400 (2008); Horvath etal., J Bacteriol 190, 1401-1412 (2008)) and Staphyloccocus aureus(SaCas9) (Hsu et al., Cell 157, 1262-1278 (2014); Ran et al., Nature520, 186-191 (2015)), might also function in our bacterial selectionassays. While the PAM of St1Cas9 has previously been characterized asNNAGAA (SEQ ID NO:3) (Esvelt et al., Nat Methods 10, 1116-1121 (2013);Fonfara et al., Nucleic Acids Res 42, 2577-2590 (2014); Deveau et al., JBacteriol 190, 1390-1400 (2008); Horvath et al., J Bacteriol 190,1401-1412 (2008)), our attempts to bioinformatically derive the SaCas9PAM using a previously described approach (Fonfara et al., Nucleic AcidsRes 42, 2577-2590 (2014)) failed to yield a consensus sequence (data notshown). Therefore, we used our site-depletion assay to determine the PAMfor SaCas9 and, as a positive control, for St1Cas9. These experimentswere performed using the two different protospacers and sgRNAs with twodifferent complementarity lengths for each protospacer, resulting infour selections for each Cas9. For St1Cas9, we identified two novel PAMsin addition to the six PAMs that had been previously described (Esveltet al., Nat Methods 10, 1116-1121 (2013); Fonfara et al., Nucleic AcidsRes 42, 2577-2590 (2014); Horvath et al., J Bacteriol 190, 1401-1412(2008)) (FIG. 4A and FIGS. 19C and 19 d, consistent with a recentdefinition of SaCas9 PAM specificity (Ran et al., Nature 520, 186-191(2015))). For SaCas9, there was PPDV variability among the fourselections mainly due to the restricted PAM preferences observed withone protospacer. As a result, only three PAMs were depleted greater than5-fold in all four experiments: NNGGGT (SEQ ID NO:4), NNGAAT (SEQ IDNO:6), NNGAGT (SEQ ID NO:5) (FIG. 4B). We did, however, identify manymore targetable PAMs with the second protospacer library, implying thatSaCas9 might recognize numerous additional PAMs (FIGS. 18C and 18D).Using PAMs identified in our site-depletion experiments (NNAGAA (SEQ IDNO:3) for St1Cas9 and NNGAGT (SEQ ID NO:5) for SaCas9), we found thatboth St1Cas9 and SaCas9 can function efficiently in the bacterialpositive selection system (FIG. 4C), suggesting that their PAMspecificities could be modified by mutagenesis and selection.

Because not all Cas9 orthologues function efficiently outside of theirnative context (Esvelt et al., Nat Methods 10, 1116-1121 (2013)), wetested whether St1Cas9 and SaCas9 can robustly cleave target sites inhuman cells. St1Cas9 has been previously shown to function as a nucleasein human cells but on only a few sites (Esvelt et al., 2013; Cong etal., Science 339, 819-823 (2013)). We assessed St1Cas9 activity on sitesharboring NNAGAA (SEQ ID NO:3) PAMs using sgRNAs with variable-lengthcomplementarity regions and found high activity at three of the fivetarget sites (FIG. 4D). For SaCas9, we observed efficient activity ateight sites harboring NNGGGT (SEQ ID NO:4) or NNGAGT (SEQ ID NO:5) PAMs(FIG. 4E). For both St1Cas9 and SaCas9 no obvious correlation betweenactivity and length of spacer complementarity was observed (FIG. 19E).We next determined whether St1Cas9 and SaCas9 could efficiently modifyendogenous loci in human cells. For St1Cas9, 7 out of 11 sites across 4genes were disrupted efficiently as judged by T7E1 assay (1 to 25%, meanof 13%; FIG. 4F), while SaCas9 displayed somewhat more robust activitiesat 16 sites tested across 4 genes (1% to 37%, mean of 19%; FIG. 4G).Once again, no distinct trend was observed when considering sgRNA spacerlength for St1Cas9 and SaCas9 (FIG. 19F). Collectively, our results showthat St1Cas9 and SaCas9 function robustly both in our bacterial-basedselection and in human cells, making them attractive candidates forengineering additional SpCas9 variants with novel PAM specificities.

TABLE 3 SEQ ID NO: Wild-type SpCas9 sequence from K1097-D1368 of SEQ IDNO: 1KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIaaMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS1097-1368HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFof SEQ TLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD ID NO: 1Selected mutant clones for VQR and EQR variant, sequence fromK1097-D1368KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI431.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI432.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYISTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKFKKLKSVKELLGITI433.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI434.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI435.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQRGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI436.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDNPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI437.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTPIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI438.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI439.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTKLGAPAAIKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI440.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLEATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI441.MERSSFEKNPMDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVEAKVEKGKSKKLKSVKELLGITI442.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFESPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI443.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPFKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPSAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESIFPKRNSDKLIARKKDWDPKKYGGLYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI444.MERSSFEKNPIDFLEAKGYKEIKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI445.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDKEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI446.MERSSFEKNPIDFLEAKGYKEVKEDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPICEQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVENRKSKKLKSVKELLGITI447.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDATIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVANVEKGKSKKLKSVKELLGITI448.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILTDANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI449.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSLEDNEQKQLFVEQHRHYLDEIIEQISEFSKRVILADANLDKVLSAYNKYRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKVWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI450.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSILVVAKVEKGKSKKLKSVKELLGITI451.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGRFSKESILPKRNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI452.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI453.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIHEQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI454.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI455.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRNKPIREQAENIIHLFTLTNLGAPAAFKYFDTMIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLVGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSRKLKSVKELLGITI456.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI457.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSLLGGDKTEVQTGGFSKESILPNRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI458.MERSSFEKKPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVKKGKSKKLKSVKELLGITI459.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI460.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDETEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAFSVLVVAKVEKGKSKKLKSVKELLGITI461.MERSFFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKDLLGITI462.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILVDANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT463.NMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAEELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDATIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI464.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIYRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVANVEKGKSKKLKSVKELLGITI465.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTDLGAPAAFKYFDTTIDRKQYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Selected mutantclones for VRER variant, sequence from K1097-D1368KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI466.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQFFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEMQTGGFSKESVLPKRNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKRLKSVKELLGI467.TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSPDDNEQKRLFVEQHKHYLDEIIEQISEFSKRVILADANRDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI468.MERSSFEENPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPKDNEQKQLFVEKHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVANVEKGKSKKLKSVKELLGITI469.MERSSYEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI470.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSFTGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI471.MERSSFEKNPIDFLEAKGYKEVKKDLLIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKDEKGKSKKLKSVKELLGITI472.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPICEQAENIIHLFTLTKLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKVIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI473.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITI474.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI475.MERSSFEKNPIDFLEAKGYKEIKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKTIREQAENTIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI476.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHHSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI477.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADGNLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI478.MERSSFEKNPIDFLEAKGYKEVKKDLLIKLPKYNLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI479.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPDYNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPKVAYSVLVVAKVEKGKSKKLKSVKELLGITI480.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDMSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI481.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLIAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEEQTGGFSKESIHPKRNSDKLIARKKDWDPKKYGGFHSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI482.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNMHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLEGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI483.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQMQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGRFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI484.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKVKSVKELLGITI485.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI486.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHFEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTMIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI487.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQPKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI488.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEIALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTKIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI489.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI490.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI491.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI492.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI493.MERSSFEKNPFDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADPNLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFLSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI494.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI495.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVIELLGITI496.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI497.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKTKKLKSVKELLGITI498.MERSSFEKNPIDFLEAKGYKEVIKDFIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSPKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI499.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVPVVAKVEKGKSKKLKSVKELLGITI500.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELESGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI501.MERSSFEKNPIDFLEAKGYKEVNKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKTYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI502.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIHEQAENIIHLFTLTNLGAPAAFKYFDTTIDRKTYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI503.MERSSFEKNPIDFLEAKGYKEVKKDLMIKLPKYSLFELKNGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTKLGAPAAFKYFDTTIDRKTYRSTKEVLDATLIHHSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI504.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKTYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGLYSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI505.MERSSFEKNPIDFLEAKGYKEVKRDLIIKLPKYSLFELKNGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKTYRSTKEVVDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESIHPKRNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI506.MERSSFEKNPIDFLEAKGYKEVKKDLIITLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADSNLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKTYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI507.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKTYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKDLLGITI508.MERSSFEKNPIDFLEAKGYKEVKKDLMIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIVHLFTLTNLGAPAAFKYFDTTIDRKTYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI509.MERSSFEKNPIDFLEAKGYKEIKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADVNLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKTYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDKTEVQTGGFSKESIHPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI510.MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKDRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKTYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD

Example 2. Engineering the PAM Specificity of Staphylococcus aureus Cas9

Because we knew what residues of Streptococcus pyogenes Cas9 (SpCas9)were important for PAM recognition (R1333 and R1335), we generated analignment of Cas9 orthologues to look for homologous residues in thePAM-interacting domain (PI domain) of Staphylococcus aureus Cas9(SaCas9) (see FIG. 6). We and others have previously shown that the PAMof SaCas9 is NNGRRT (SEQ ID NO:46) (where N is any nucleotide, and R isan A or G). The preference for a G at the 3^(rd) position of the PAMappeared to be the most strict requirement based on our data, so wehypothesized that positively charged residues like lysine (K) orarginine (R) might be mediating that interaction. As shown in FIG. 6,there are a number of candidate residues in SaCas9 in the homologousregion to R1333 and R1335 of SpCas9, including K1101, R1012, R1015,K1018, and K1023.

We generated alanine (A) and glutamine (Q) substitutions at these fivepositions to determine if the mutant clones could still cleave a sitecontaining the canonical NNGRRT PAM (SEQ ID NO:46), or possibly cleavethe previously non-targetable PAM of NNARRT (SEQ ID NO:43) (FIG. 7). Weutilized our bacterial assay (described in the previous patentapplication), where activity of Cas9 can be visualized by survival ofbacterial colonies when plated under a selective condition. The relativeactivity of Cas9 can be quantified by calculating the ratio of bacterialcolonies growing on the selective versus the non-selective media. InFIG. 7, we show that only the R1015A and R1015Q mutations affect theability of SaCas9 to recognize a canonical NNGAGT (SEQ ID NO:5) PAM,while no mutations enable targeting of NNARRT (SEQ ID NO:43) PAMs(NNAAGT (SEQ ID NO:41) or NNAGGT (SEQ ID NO:42)). These resultssuggested to us that R1015 plays a role in PAM recognition by SaCas9.

We then selected randomly mutagenized either wild-type SaCas9, or theR1015Q variant and selected for altered PAM specificity clones againstsites containing NNAAGT (SEQ ID NO:41) or NNAGGT (SEQ ID NO:42) PAMs (aspreviously described for SpCas9). We identified, re-screened, andsequenced a number of mutant clones that could target these PAMs, withtheir amino acid sequences shown in FIG. 8 (and Table 6). In summary ofthese sequences, a number of changes appear to be very important foraltering SaCas9 specificity (R1015Q, R1015H, E782K), while many othermutations may also contribute (N968K, E735K, K929R, A1021T, K1044N).

After identifying the positions and mutations essential for altering thePAM specificity of SaCas9 to NNARRT (SEQ ID NO:43), we assessed thecontributions of the most abundant mutations to the specificity changeby making single, double, and triple mutants combinations (Table 5).When testing these mutations against various PAMs in our positiveselection (as previously described), we observed that a number ofmutations allowed activity on both a canonical NNGAGT (SEQ ID NO:5) andnon-canonical NNAAGT (SEQ ID NO:41) or NNAGGT (SEQ ID NO:42) PAMs,whereas the wild-type SaCas9 enzyme had very low activity on thenon-canonical PAMs. Specifically, it appeared as though the triplemutations enabled a relaxed specificity at the third position of the PAM(KKQ, KKH, GKQ, GKH—named based on mutations to positionsE782/N968/R1015), leading to a consensus PAM motif of NNRRRT (SEQ IDNO:45) versus the canonical NNGRRT (SEQ ID NO:46). This relaxation ofthe PAM requirement theoretically doubles the targeting range of SpCas9.Henceforth, variants will be named based on their identities atpositions 782, 968, and 1015. For example, E782K/N968K/R1015H would benamed the SaCas9 KKH variant.

TABLE 5 SaCas9 mutant activity in the bacterial screen NNGAGT NNAAGTNNAGGT (SEQ ID (SEQ ID (SEQ ID mutation(s) NO: 5) NO: 41) NO: 42) E782N968 R1015 % activity % activity % activity 100.0 21.4 15.7 Q 0.0 4.30.0 H 100.0 100.0 57.1 K 85.7 61.4 57.1 G 85.7 57.1 57.1 K 100.0 57.157.1 K Q 85.7 92.9 85.7 K H 100.0 100.0 85.7 G Q 71.4 85.7 71.4 G H100.0 85.7 85.7 K Q 85.7 85.7 85.7 K H 85.7 92.9 92.9 K K 71.4 71.4 71.4G K 85.7 71.4 71.4 K K Q 100.0 100.0 100.0 K K H 92.9 100.0 100.0 G K Q92.9 92.9 100.0 G K H 100.0 100.0 100.0

We next assessed two of the triple mutants in the human cell EGFPdisruption assay (as previously described) to determine whether theengineered variants could target non-canonical PAMs in a human cellcontext (FIG. 9). Variants capable of targeting sites within the EGFPgene containing non-canonical PAMs will disrupt the EGFP coding frame,leading to loss of signal. The results revealed that both the KKQ andKKH mutants retained similar activity to wild-type SaCas9 on canonicalNNGRRT (SEQ ID NO:46) PAMs, but had much higher activity on NNARRT (SEQID NO:43) PAMs.

Overall, we've identified mutations in SaCas9 (KKQ or KKH variants) thatappear to relax the preference of the wild-type enzyme at the thirdposition of the PAM from a G to an R (A or G). This effectively relaxesthe targeting of SaCas9 from an NNGRRT (SEQ ID NO:46) PAM constraint toan NNRRRT (SEQ ID NO:45) PAM.

Because we had successfully derived variants that could target NNARRT(SEQ ID NO:43) PAMs in human cells, we next asked the question ofwhether we could engineer variants with specificity for NNCRRT (SEQ IDNO:47) or NNTRRT (SEQ ID NO:48). To do so, we first mutated R1015 to E(in the case of specifying a C at the 3^(rd) position of the PAM) and toL or M (in the case of specifying a T at the 3^(rd) position of thePAM), and tested these against their expected PAMs in our bacterialpositive selection assay (previously described) (FIG. 10). We observedthat wild-type SaCas9 could inefficiently cleave a site containing anNNCAGT (SEQ ID NO:511) PAM, that an R1015E variant had slightly betteractivity on the same site, and that wild-type or any of the otherdirected mutations did not convey activity against other PAMs (FIG. 10).This suggested that as we saw with R1015Q, other mutations would benecessary to engineer SaCas9 variants that could target NNCRRT (SEQ IDNO:47) and NNTRRT (SEQ ID NO:48) PAMs.

For the SaCas9 evolved variants against NNARRT (SEQ ID NO:43) PAMs, theE782K and N968K mutations were necessary and essential along with theR1015(H/Q). To test whether these mutations would increase the activityof the R1015(E/L/M) variants against their expected PAM, we generatedthe KKE, KKL, and KKM variants. As shown in FIG. 11, the KKE, KKL, andKKM all had robust activity against their expected PAMs.

We were also curious as to whether the KKQ, KKH, KKE, KKL, or KKMvariants had relaxed specificity against any nucleotide at the 3^(rd)position of the PAM, so we interrogated a number of sites in ourbacterial positive selection assay containing NNNRRT PAMs. As shown inFIG. 11, with a few exceptions nearly all of these variants can cleaveall sites tested that contain NNNRRT PAMs. This indicated that they hada relaxed specificity at the 3^(rd) position of the PAM as they canefficiently target NNNRRT sites. This is in contrast to the wild-typeprotein (ENR) that can only efficiently target the NNGAGT (SEQ ID NO:5)site, with very low activity on a few NNNRRT sites. In summary, the KKH(and other similar derivatives shown in FIG. 11) variant can targetsites containing NNNRRT PAMs in bacteria, effectively quadrupling thetargeting range of SaCas9.

Thus, the KKH variant (and some of the other variants in FIG. 6) cantarget NNNRRT PAMs in bacteria, effectively quadrupling the targetingrange of SaCas9.

TABLE 6 SEQ ID NO: residues A652-G1053 of SaCas9 Wild Type SaCas9ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN AaADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYK 652-1053YSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMY ofHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNA SEQ IDHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEE NO: 2.AKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG Sequences of selected clonesof SaCas9 variantsATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 53.ADFIFKEWKRLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVKSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSNKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 54.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKELINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNMVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFMASFYKNDLIKFNGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDIKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANA 55.DFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLNITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFISSFYSNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKRIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 56.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIVITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPEIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 57.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIRINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSIKGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 58.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDFKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDNYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYRGYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 59.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMKDKRPPHIIKTIASKTQSIIKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 60.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEHEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSNKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 61.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHINDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKRPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 62.ADFIFKEWKKLDKAKKLMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVRNLDVIKKENHYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 63.ADFIFKEWKKLDKAKKVMENQMFEEKQAESKPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNTKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIVKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 64.ADFIFKEWKKLDRAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGYTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTITSKTQSIKKYSTDILGNLYEVKSKKQPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 65.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLILEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 66.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGIYKFVTVKNMDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYIENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 67.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVRNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQVIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 68.ADFIFKEWKRLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDDKNPLYKYYEETGNYLIKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVRNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIAYFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK*GATRGLMNLLRSYFRVNNLDVKVKSINGGFTRFLRRKWKFKKERNKGYKHHAEDALIIAN 69.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQITKKGATRGLMNLLRNYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 70.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDQQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKNENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQNIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQII*KGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 71.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGYYLTKYSKKDNGPVIKKIKYYGNKINAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIVKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 72.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 73.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 74.ADFIFKEWKKLDKAKKLMENQMFEEKQAESMPEIETEQEYKEIFMTPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYHEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 75. andADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYK 966YSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDIGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNYKRPPQIIKTIASKTQSIKKYSSDILGNLYEVKSKKHP*IIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 76.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIINTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 77.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKELFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKCSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 78.ADFIFKEWKKLDKAKKVMENQMFEKKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRGLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEDTGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDLIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENVNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 79.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGIYKFVTVKNLDVIKKENYYEVNSKCYEKAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSNKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 80.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFIIPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 81.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETQQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASYYNNDLIKINGELYRVIGVNNDLLNRIEVKMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPHIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 82.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNILNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIRYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 83.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYFENMNVKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 84.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKHNRELVNDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTITSKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 85.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIMDFKDYKYSHRVDKKPNRELINDTLYSTRKDEKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVRNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 86.ADFIFKEWKKLDKAKKVMENQMFEEKQAVSMPEIETEQEYKEIFINPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKYNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSRKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYRENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 87.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKNENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENINGKRPPQIIKTITSKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNNGYKHHAEDALIIAN 88.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKEFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGIYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYGEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLEIMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSNKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 89.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKVIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYIYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNVYEVKSKKHPQIIIKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 90.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETGQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLKSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 91.ADFIFKEWKKLDKSKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKHNRKLINDTLYSTRKDDKGNTLIVNNINGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNTIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKPKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 92. andADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYK 966YSHRVDKKPNRKLINDTLYSTREDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDISDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIYITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHP*IIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 93.ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPYQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVRNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 94.ADFIFKEWKKLDNAKKVMENQMFEEKRAESMPEIETEQEYKEIFITPHQIKHIKDFKDFKYSHMVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLIYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 95. andADFIFKEWIRLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYK 966YSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHP*IIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIVAN 96. andADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYK 966YSHRVDKKPNRELINDTLYSTRKDDKGNTLIVINLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHP*IIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 97. andADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYK 966YSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHP*IIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 98. andADFIFKEWKKLDKAKKVMENQMFEEKQAMSMPEIETEQEYKEIFITPHQIKHIKDFKDY 966KYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPDSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSQKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 99.ADFIFKEWKKLDKAKKVMENQMFEEKQAGSMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNRLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLESMNDKRPPQIIKTIASKTQTIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYYRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 100. ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLTNKSPGKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAYLDITDDYPNSRNNVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIEKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 101. ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKFKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKDNYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIATKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRTYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 102. ADFIFKEWKKLDKAKKVMENQMFEEKHAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLIDKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK*GATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 103. ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYNEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLYVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIAN 104. ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPQIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG

Methods for Example 3

The following materials and methods were used in Example 3.

Plasmids and Oligonucleotides

Oligonucleotides are listed in Table 11, sgRNA target sites are listedin Table 12, and plasmids used in this study are listed in Table 10.

Bacterial Cas9/sgRNA expression plasmids were used to express both ahuman codon optimized version of SaCas9 and the sgRNA, each expressedunder a separate T7 promoter. Bacterial expression plasmids used in theselections were derived from BPK2101 (see Examples 1-2) while those usedin the site-depletion assay were modified to express a sgRNA with ashortened repeat:anti-repeat sequence (see below). All sgRNAs in thesebacterial expression plasmids included two guanines at the 5′ end of thespacer sequence for proper expression from the T7 promoter.

To generate libraries of SaCas9 variants, amino acids M657-G1053 ofSaCas9 were randomly mutagenized using Mutazyme II (AgilentTechnologies) at a frequency of ˜5.5 mutations/kilobase. Both wild-typeand R1015Q SaCas9 were used as starting template for mutagenesis,resulting in two libraries with estimated complexities of greater than6×10⁶ clones.

Positive selection plasmids were assembled by ligating oligonucleotideduplexes encoding target sites into XbarSphI-digested p11-lacY-wtx1(Chen, Z. & Zhao, H. A highly sensitive selection method for directedevolution of homing endonucleases. Nucleic Acids Res 33, e154 (2005)).For the site-depletion experiments, two separate libraries containingdifferent spacer sequences were generated. For each library, anoligonucleotide containing 8 randomized nucleotides adjacent to thespacer sequence (in place of the PAM) was complexed with a bottom strandprimer and filled in using Klenow(-exo) (refer to Table 11). Theresulting product was digested with EcoRI and ligated intoEcoRI/SphI-digested p11-lacY-wtx1. Estimated complexities of the twosite-depletion libraries were greater than 4×10⁶ clones.

For human cell experiments, human codon-optimized wild-type and variantSaCas9s were expressed from a plasmid containing a CAG promoter (Table12). sgRNA expression plasmids (containing a U6 promoter) were generatedby ligating oligonucleotide duplexes encoding the spacer sequence intoBsmBI digested VVT1 (See Examples 1-2 or BPK2660 (containing the fulllength 120 nt crRNA:tracrRNA sgRNA or a 84 nt shortenedrepeat:anti-repeat version, respectively). All sgRNAs used in this studyfor human expression included one guanine at the 5′ end of the spacer toensure proper expression from the U6 promoter, and also used a shortenedsgRNA (FIG. 37A-B) similar to that previously described (Ran, F. A. etal. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520,186-191 (2015)).

Bioinformatic Analysis of Cas9 Orthologue Sequences

Similar to alignments performed in previous studies (Fonfara, I. et al.Phylogeny of Cas9 determines functional exchangeability of dual-RNA andCas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res 42,2577-2590 (2014); Ran, F. A. et al. In vivo genome editing usingStaphylococcus aureus Cas9. Nature 520, 186-191 (2015); Anders, C.,Niewoehner, O., Duerst, A. & Jinek, M. Structural basis of PAM-dependenttarget DNA recognition by the Cas9 endonuclease. Nature 513, 569-573(2014)), Cas9 orthologues similar to both SpCas9 and SaCas9 were alignedusing ClustalW2 (ebi.ac.uk/Tools/msa/clustalw2/). The resultingphylogenetic tree and protein alignment were visualized using Geneiousversion 8.1.6 and ESPript (espript.ibcp.fr/ESPript/ESPript/).

Bacterial-Based Positive Selection Assay

The bacterial positive selection assays were performed as previouslydescribed (See Examples 1-2). Briefly, Cas9/sgRNA plasmids weretransformed into E. coli BW25141(λDE3) (Kleinstiver et al., NucleicAcids Res 38, 2411-2427 (2010)) containing a positive selection plasmid.Transformations were plated on both non-selective (chloramphenicol) andselective (chloramphenicol+10 mM arabinose) conditions. Cas9 cleavage ofthe selection plasmid was estimated by calculating the percent survival:(# of colonies on selective plates/# of colonies on non-selectiveplates)×100. To select for SaCas9 variants capable of recognizingalternative PAMs, the wild-type and R1015Q libraries with mutagenized PIdomains were transformed into competent E. coli BW25141(λDE3) containingpositive selection plasmids with NNAAGT (SEQ ID NO:41), NNAGGT (SEQ IDNO:42), NNCAGT (SEQ ID NO:511), NNCGGT (SEQ ID NO:512), NNTAGT (SEQ IDNO:513), or NNTGGT (SEQ ID NO:514) PAMs. Approximately 1×10⁵ clones werescreened by plating on selective conditions, and surviving coloniescontaining SaCas9 variants presumed to cleave the selection plasmid weremini-prepped (MGH DNA Core). All variants were re-screened individuallyin the positive selection assay, and those with greater than ˜20%survival were sequenced to determine the mutations required forrecognition of the alternate PAM.

Bacterial-Based Site-Depletion Assay

The site-depletion experiments were performed as previously described(See Examples 1-2). Briefly, the randomized PAM libraries wereelectroporated into competent E. coli BW25141(λDE3) containing eitherwild-type, catalytically inactive (D10A/H557A), or KKH variantSaCas9/sgRNA plasmids. Greater than 1×10⁵ colonies were plated onchloramphenicol/carbenicillin plates, and selection plasmids with PAMsresistant to Cas9 targeting contained within the surviving colonies wereisolated by maxiprep (Qiagen). The region of the plasmid containing thespacer sequence and PAM was PCR-amplified using the primers listed inTable 11. The KAPA HTP library preparation kit (KAPA BioSystems) wasused to generate a dual-indexed Tru-seq Illumina sequencing libraryusing ˜500 ng purified PCR product from each site-depletion conditionprior to an Illumina MiSeq high-throughput sequencing run at theDana-Farber Cancer Institute Molecular Biology Core. The data from thesite-depletion experiments was analyzed as previously described (SeeExamples 1-2), with the exception that the script was modified toanalyze 8 randomized nucleotides. Cas9 ability to recognize PAMs wasdetermined by calculating the post-selection PAM depletion value (PPDV)of any given PAM: the ratio of the post-selection frequency of that PAMto the pre-selection library frequency. A control experiment usingcatalytically inactive SaCas9 was used to establish that a PPDV of 0.794represents statistically significant depletion relative to the inputlibrary.

Human Cell Culture and Transfection

U2OS cells obtained from our collaborator T. Cathomen (Freiburg) andU2OS.EGFP cells harboring a single integrated copy of an EGFP-PESTreporter gene (Reyon, D. et al. FLASH assembly of TALENs forhigh-throughput genome editing. Nat Biotechnol 30, 460-465 (2012)) werecultured in Advanced DMEM medium (Life Technologies) with 10% FBS,penicillin/streptomycin, and 2 mM GlutaMAX (Life Technologies) at 37° C.with 5% CO₂. Cell line identities were validated by STR profiling (ATCC)and deep sequencing, and cells were tested bi-weekly for mycoplasmacontamination. U2OS.EGFP culture medium was additionally supplementedwith 400 μg/mL G418. Cells were co-transfected with 750 ng Cas9 plasmidand 250 ng sgRNA plasmid using the DN-100 program of a Lonza4D-nucleofector following the manufacturer's instructions.

Human Cell EGFP Disruption Assay

EGFP disruption experiments were performed as previously described (Fu,Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Casnucleases in human cells. Nat Biotechnol 31, 822-826 (2013); Reyon, D.et al. FLASH assembly of TALENs for high-throughput genome editing. NatBiotechnol 30, 460-465 (2012)). Approximately 52 hourspost-transfection, a Fortessa flow cytometer (BD Biosciences) was usedto measure EGFP fluorescence in transfected U2OS.EGFP cells. Negativecontrol transfections of Cas9 and empty U6 promoter plasmids were usedto establish background EGFP loss at ˜2.5% for all experiments(represented as a red dashed lined in FIGs).

T7E1 Assay

T7E1 assays were performed as previously described (Reyon, D. et al.FLASH assembly of TALENs for high-throughput genome editing. NatBiotechnol 30, 460-465 (2012)) to quantify Cas9-induced mutagenesis atendogenous loci in human cells. Approximately 72 hourspost-transfection, genomic DNA was isolated using the AgencourtDNAdvance Genomic DNA Isolation Kit (Beckman Coulter Genomics). Targetloci were PCR-amplified from ˜100 ng of genomic DNA using the primerslisted in Table 11. Following an Agencourt Ampure XP clean-up step(Beckman Coulter Genomics), ˜200 ng purified PCR product was denaturedand hybridized prior to digestion with T7E1 (New England Biolabs).Following a second clean-up step, mutagenesis frequencies werequantified using a Qiaxcel capillary electrophoresis instrument(Qiagen).

GUIDE-Seq Experiments

GUIDE-seq experiments were performed and analyzed as previouslydescribed (Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling ofoff-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33, 187-197(2015)). Briefly, U2OS cells were transfected as described above withCas9 and sgRNA plasmids, as well as 100 pmol of a phosphorylated,phosphorothioate-modified double-stranded oligodeoxynucleotide (dsODN)with an embedded NdeI site. Restriction fragment length polymorphism(RFLP) analyses were performed to determine frequency of dsODN-tagintegration frequencies ((See Examples 1-2; Tsai, S. Q. et al. GUIDE-seqenables genome-wide profiling of off-target cleavage by CRISPR-Casnucleases. Nat Biotechnol 33, 187-197 (2015)), and T7E1 assays wereperformed to quantify on-target Cas9 mutagenesis frequencies. dsODNtag-specific amplification and library preparation (Tsai, S. Q. et al.GUIDE-seq enables genome-wide profiling of off-target cleavage byCRISPR-Cas nucleases. Nat Biotechnol 33, 187-197 (2015)) was performedprior to high-throughput sequencing using an Illumina MiSeq Sequencer.When mapping potential off-target sites, the cut-off for alignment tothe on-target spacer sequence was set at 8 mismatches for 21 nucleotidespacers, 9 mismatches for 22 nucleotide spacers, and 10 mismatches for23 nucleotide spacers. Off-target sites with potential DNA- orRNA-bulges (Lin, Y. et al. CRISPR/Cas9 systems have off-target activitywith insertions or deletions between target DNA and guide RNA sequences.Nucleic Acids Res 42, 7473-7485 (2014)) were identified by manualalignment.

TABLE 10 Plasmids used in Example 3 SEQ Name ID NO: Description BPK210110 T7-humanSaCas9-NLS-3xFLAG-T7-Bsalcassette-Sa-sgRNA(120) Addgene ID:65770 T7 promoters at 1-17 and 3418-3434, human codon optimized S.aureus Cas9 at 88- 3352, NLS at 3256-3276, 3xFLAG tag at 3283-3348, Bsalsites at 3437-3442 and 3485- 3490, gRNA at 3492-3616, T7 terminator at3627-2674 of SEQ ID NO: 10. MSP2283 21T7-humanSaCas9-NLS-3xFLAG-T7-site1-Sa-sgRNA(84) T7 promoters at nts 1-17and 3418-3434, human codon optimized S. aureus Cas9 at 88- 3351, NLS at3256-3276, 3xFLAG tag at 3243-33348, site 1 spacer at 3435-3455,sgRNA(84) at 3456-3539, T7 terminator at 3562-3609 of SEQ ID NO: 21MSP2262 22 T7-humanSadCas9(D10A, H557A)-NLS-3xFLAG-T7-site1-Sa-sgRNA(84)T7 promoters at nts 1-17 and 3418-3434, human codon optimized S. aureusCas9 at 88- 3351, modified codons at 118-120 and 1759-1761, NLS at3256-3276, 3xFLAG tag at 3243-33348, site 1 spacer at 3435-3455,sgRNA(84) at 3456-3539, T7 terminator at 3562-3609 of SEQ ID NO: 22MSP2253 23 T7-humanSaCas9(E782K, N968K,R1015H)-NLS-3xFLAG-T7-site1-Sa-sgRNA(84) T7 promoters at nts 1-17 and3418-3434, human codon optimized S. aureus Cas9 at 88- 3351, modifiedcodons at 2434-2436, 2992-2994, and 3133-3135, NLS at 3256-3276, 3xFLAGtag at 3243-33348, site 1 spacer at 3435-3455, sgRNA(84) at 3456-3539,T7 terminator at 3562-3609 of SEQ ID NO: 23 MSP2266 24T7-humanSaCas9-NLS-3xFLAG-T7-site2-Sa-sgRNA(84) T7 promoters at 1-17 and3419-3434, human codon optimized S. aureus Cas9 at 88- 3351, NLS at3256-3276, 3xFLAG tag at 3283-3348, site 2 spacer at 3435-3455,sgRNA(84) at 3456-3539, T7 terminator at 3562-3609 of SEQ ID NO: 24MSP2279 25 T7-humanSadCas9(D10A, H557A)-NLS-3xFLAG-T7-site2-Sa-sgRNA(84)T7 promoters at 1-17 and 3419-3434, human codon optimized S. aureus Cas9at 88- 3351, modified codons at 118-120 and 1759-1761, NLS at 3256-3276,3xFLAG tag at 3283-3348, site 2 spacer at 3435-3455, sgRNA(84) at3456-3539, T7 terminator at 3562-3609 of SEQ ID NO: 25 MSP2292 26T7-humanSaCas9(E782K, N968K, R1015H)-NLS-3xFLAG-T7-site2-Sa-sgRNA(84) T7promoters at 1-17 and 3419-3434, human codon optimized S. aureus Cas9 at88- 3351, modified codons at 2434-2436, 2992-2994, and 3133-3135, NLS at3256-3276, 3xFLAG tag at 3283-3348, site 2 spacer at 3435-3455,sgRNA(84) at 3456-3539, T7 terminator at 3562-3609 of SEQ ID NO: 26p11-lacY- — BAD-ccDB-Amp^(R)-AraC-lacY(A177C) (Chen et al, 2005) wtx1BPK2139 17 CAG-humanSaCas9-NLS-3xFLAG Addgene ID: 65776 Human codonoptimized S. aureus Cas9 1-3195, NLS 3169-3189, 3xFLAG tag 3196- 3261 ofSEQ ID NO: 17. MSP1830 27 CAG-humanSaCas9(E782K, N968K,R1015H)-NLS-3xFLAG (KKH variant) Human codon optimized S. aureus Cas91-3264, NLS 3169-3189, modified codons at 2347-2349, 2905-2907, and3046-3048, 3xFLAG tag 3196-3261 of SEQ ID NO: 27 VVT1 20U6-BsmBlcassette-Sa-sgRNA(120) Addgene ID: 65779 U6 promoter 1-318,BsmBl sites at 320-325 and 333-338, S. aureus gRNA 340-466, U6terminator 459-466 of SEQ ID NO: 20. BPK2660 28U6-BsmBlcassette-Sa-sgRNA(84) U6 promoter 1-318, BsmBl sites at 320-325and 333-338, S. aureus gRNA 340-423, U6 terminator 424-430 of SEQ ID NO:28.

TABLE 11 Oligonucleotides used in Example 3 SEQ ID Sequence DescriptionNO: Oligos used to generate positive selection plasmidsctagaGGGtGGGcGGGaGGGTCGCCCTCGAACTTCAC top oligo to clone site 2 with anNNGAGT (SEQ ID NO: 5) 515 CTtgGAGTgcatg PAM into the positive selectionvector (Xbal/Sphl cut p11-lacY-wtx1)cACTCcaAGGTGAAGTTCGAGGGCGACCCtCCCgCCC bottom oligo to clone site 2 intothe positive selection 516 aCCCt vector Oligos used to generatelibraries for site-depletion experimentsGCAGgaattcGGGAGGGGCACGGGCAGCTTGCCGGN top strand oligo for site 1 PAMlibrary, cut with EcoRI 517 NNNNNNNCTNNNGCGCAGGTCACGAGGCATG once filledin GCAGgaattcGGAGGGTCGCCCTCGAACTTCACCTNN top strand oligo for site 2 PAMlibrary, cut with EcoRI 518 NNNNNNCTNNNGCGCAGGTCACGAGGCATG once filledin /5Phos/CCTCGTGACCTGCGC reverse primer to fill in library oligos 200Primers used to amplify site-depletion libraries for sequencingGATACCGCTCGCCGCAGC forward primer 201 CTGCGTTCTGATTTAATCTGTATCAGGCreverse primer 202 Primers used for T7E1 and RFLP experimentsGGAGCAGCTGGTCAGAGGGG forward primer targeted to EMX1 in U2OS human cells209 CCATAGGGAAGGGGGACACTGG reverse primer targeted to EMX1 in U2OS humancells 210 GGGCCGGGAAAGAGTTGCTG forward primer targeted to FANCF in U2OShuman cells 211 GCCCTACATCTGCTCTCCCTCC reverse primer targeted to FANCFin U2OS human cells 212 CCAGCACAACTTACTCGCACTTGAC forward primertargeted to RUNX1 in U2OS human cells 213 CATCACCAACCCACAGCCAAGG reverseprimer targeted to RUNX1 in U2OS human cells 214 TCCAGATGGCACATTGTGAGforward primer targeted to VEGFA in U2OS human cells 652AGGGAGCAGGAAAGTGAGGT reverse primer targeted to VEGFA in U2OS humancells 653

TABLE 12 sgRNA target sites for Example 3 In VVT1 (120) EGFP Spacer SEQSEQ length ID ID Prep Name Name (nt) Spacer Sequence NO:: Sequence withPAM NO: MSP1428 NNGRRT 21 GCCCTCGAACTTCACCTCGGC 405GCCCTCGAACTTCACCTCGGCGCGGGT 406 1 (SEQ ID NO: 46) MSP1400 NNGRRT 21GCAACATCCTGGGGCACAAGC 397 GCAACATCCTGGGGCACAAGCTGGAGT 398 2 (SEQ ID NO:46) MSP1401 NNGRRT 21 GTTGTACTCCAGCTTGTGCCC 519GTTGTACTCCAGCTTGTGCCCCAGGAT 520 3 (SEQ ID NO: 46) MSP1403 NNGRRT 22GCAAGGGCGAGGAGCTGTTCAC 409 GCAAGGGCGAGGAGCTGTTCACCGGGGT 410 4 (SEQ IDNO: 46) MSP1748 NNARRT 20 GGACGGCGACGTAAACGGCC 521GGACGGCGACGTAAACGGCCACAAGT 522 1 (SEQ ID NO: 43) MSP1754 NNARRT 21GAACTTCAGGGTCAGCTTGCC 523 GAACTTCAGGGTCAGCTTGCCGTAGGT 524 5 (SEQ ID NO:43) MSP2030 NNCRRT 20 GTCGATGCCCTTCAGCTCGA 525GTCGATGCCCTTCAGCTCGATGCGGT 526 2 (SEQ ID NO: 47) MSP2034 NNCRRT 22GTGACCACCCTGACCTACGGCG 527 GTGACCACCCTGACCTACGGCGTGCAGT 528 4 (SEQ IDNO: 47) MSP2040 NNTRRT 20 GATATAGACGTTGTGGCTGT 529GATATAGACGTTGTGGCTGTTGTAGT 530 1 (SEQ ID NO: 48) MSP2045 NNTRRT 21GGTGAAGTTCGAGGGCGACAC 531 GGTGAAGTTCGAGGGCGACACCCTGGT 532 3 (SEQ ID NO:48) In BPK2660 (84) EGFP Spacer length Prep Name Name (nt) SpacerSequence Sequence with PAM MSP2149* NNARRT 20 GGACGGCGACGTAAACGGCC 521GGACGGCGACGTAAACGGCCACAAGT 522 1 (SEQ ID NO: 43) MSP2152 NNARRT 21GTAGTTGCCGTCGTCCTTGAA 654 GTAGTTGCCGTCGTCCTTGAAGAAGAT 655 2 (SEQ ID NO:43) MSP2153 NNARRT 22 GCCACCTACGGCAAGCTGACCC 656GCCACCTACGGCAAGCTGACCCTGAAGT 657 3 (SEQ ID NO: 43) MSP2154 NNARRT 23GACGGCAACTACAAGACCCGCGC 658 GACGGCAACTACAAGACCCGCGCCGAGGT 659 4 (SEQ IDNO: 43) MSP2150* NNARRT 21 GAACTTCAGGGTCAGCTTGCC 523GAACTTCAGGGTCAGCTTGCCGTAGGT 524 5 (SEQ ID NO: 43) MSP2155 NNCRRT 20GCGTGTCCGGCGAGGGCGAG 305 GCGTGTCCGGCGAGGGCGAGGGCGAT 533 1 (SEQ ID NO:47) MSP2156* NNCRRT 20 GTCGATGCCCTTCAGCTCGA 525GTCGATGCCCTTCAGCTCGATGCGGT 526 2 (SEQ ID NO: 47) MSP2158 NNCRRT 22GCTCGACCAGGATGGGCACCAC 534 GCTCGACCAGGATGGGCACCACCCCGGT 535 3 (SEQ IDNO: 47) MSP2159* NNCRRT 22 GTGACCACCCTGACCTACGGCG 527GTGACCACCCTGACCTACGGCGTGCAGT 528 4 (SEQ ID NO: 47) MSP2145* NNGRRT 21GCCCTCGAACTTCACCTCGGC 405 GCCCTCGAACTTCACCTCGGCGCGGGT 406 1 (SEQ ID NO:46) MSP2146* NNGRRT 21 GCAACATCCTGGGGCACAAGC 397GCAACATCCTGGGGCACAAGCTGGAGT 398 2 (SEQ ID NO: 46) MSP2147 NNGRRT 21GTTGTACTCCAGCTTGTGCCC 519 GTTGTACTCCAGCTTGTGCCCCAGGAT 520 3 (SEQ ID NO:46) MSP2148 NNGRRT 22 GCAAGGGCGAGGAGCTGTTCAC 409GCAAGGGCGAGGAGCTGTTCACCGGGGT 410 4 (SEQ ID NO: 46) MSP2161* NNTRRT 20GATATAGACGTTGTGGCTGT 529 GATATAGACGTTGTGGCTGTTGTAGT 530 1 (SEQ ID NO:48) MSP2162 NNTRRT 21 GGGCGAGGAGCTGTTCACCGG 536GGGCGAGGAGCTGTTCACCGGGGTGGT 537 2 (SEQ ID NO: 48) MSP2164* NNTRRT 21GGTGAAGTTCGAGGGCGACAC 531 GGTGAAGTTCGAGGGCGACACCCTGGT 532 3 (SEQ ID NO:48) MSP2163 NNTRRT 21 GCACTGCACGCCGTAGGTCAG 538GCACTGCACGCCGTAGGTCAGGGTGGT 539 4 (SEQ ID NO: 48) Endogenous genesSpacer length Prep Name Name (nt) Spacer Sequence Sequence with PAM EMX1MSP2184** EMX1 1 22 GTGTGGTTCCAGAACCGGAGGA 540GTGTGGTTCCAGAACCGGAGGACAAAGT 541 MSP2185 EMX1 2 21 GCAGGCTCTCCGAGGAGAAGG542 GCAGGCTCTCCGAGGAGAAGGCCAAGT 543 MSP2183 EMX1 3 23GCCCCTCCCTCCCTGGCCCAGGT 544. GCCCCTCCCTCCCTGGCCCAGGTGAAGGT 545.MSP2199** EMX1 4 21 GCTCAGCCTGAGTGTTGAGGC 546.GCTCAGCCTGAGTGTTGAGGCCCCAGT 547. MSP2202 EMX1 5 21GCCTGCTTCGTGGCAATGCGCC 548. GCCTGCTTCGTGGCAATGCGCCACCGGT 549. MSP2168**EMX1 6 21 GCAACCACAAACCCACGAGGG 550. GCAACCACAAACCCACGAGGGCAGAGT 551.MSP2169 EMX1 7 21 GGCCTCCCCAAAGCCTGGCCA 552. GGCCTCCCCAAAGCCTGGCCAGGGAGT553. MSP2170 EMX1 8 23 GCAGAAGCTGGAGGAGGAAGGGC 554.GCAGAAGCTGGAGGAGGAAGGGCCTGAGT 555. MSP2201 EMX1 9 21GCTTCGTGGCAATGCGCCACCG 556. GCTTCGTGGCAATGCGCCACCGGTTGAT 557. MSP2200**EMX1 10 22 GGCTCTCCGAGGAGAAGGCCA 558. GGCTCTCCGAGGAGAAGGCCAAGTGGT 559.FANCF MSP2189 FANCF 1 22 GCCTCTCTGCAATGCTATTGGT 560.GCCTCTCTGCAATGCTATTGGTCGAAAT 561. MSP2190 FANCF 2 21GCGTACTGATTGGAACATCCG 562. GCGTACTGATTGGAACATCCGCGAAAT 563. MSP2186FANCF 3 23 GACGTCACAGTGACCGAGGGCCT 564. GACGTCACAGTGACCGAGGGCCTGGAAGT565. MSP2187 FANCF 4 23 GCCCGGCGCACGGTGGCGGGGTC 566.GCCCGGCGCACGGTGGCGGGGTCCCAGGT 567. MSP2188 FANCF 5 21GGCGGGGTCCCAGGTGCTGAC 568. GGCGGGGTCCCAGGTGCTGACGTAGGT 569. MSP2205FANCF 6 21 GGCGTATCATTTCGCGGATGT 570. GGCGTATCATTTCGCGGATGTTCCAAT 571.MSP2208 FANCF 7 22 GAGACCGCCAGAAGCTCGGAAA 572.GAGACCGCCAGAAGCTCGGAAAAGCGAT 573. MSP2204 FANCF 8 21GGATCGCTTTTCCGAGCTTCT 574. GGATCGCTTTTCCGAGCTTCTGGCGGT 575. MSP2207**FANCF 9 22 GCGCCCACTGCAAGGCCCGGCG 576. GCGCCCACTGCAAGGCCCGGCGCACGGT 577.MSP2172** FANCF 10 21 GTAGGGCCTTCGCGCACCTCA 578.GTAGGGCCTTCGCGCACCTCATGGAAT 579. MSP2174 FANCF 11 22GCAGCCGCCGCTCCAGAGCCGT 580. GCAGCCGCCGCTCCAGAGCCGTGCGAAT 581. MSP2332FANCF 12 22 GGCCATGCCGACCAAAGCGCCG 582. GGCCATGCCGACCAAAGCGCCGATGGAT583. MSP2171** FANCF 13 21 GCAAGGCCCGGCGCACGGTGG 584.GCAAGGCCCGGCGCACGGTGGCGGGGT 585. MSP2173 FANCF 14 22GAGGCAAGAGGGCGGCTTTGGG 586. GAGGCAAGAGGGCGGCTTTGGGCGGGGT 587. MSP2206FANCF 15 22 GTGACCGAGGGCCTGGAAGTTC 588. GTGACCGAGGGCCTGGAAGTTCGCTAAT589. MSP2203** FANCF 16 21 GGGGTCCCAGGTGCTGACGTA 590.GGGGTCCCAGGTGCTGACGTAGGTAGT 591. MSP2209 FANCF 17 22GTACTGATTGGAACATCCGCGA 592. GTACTGATTGGAACATCCGCGAAATGAT 593. RUNX1MSP2192 RUNX1 1 23 GTCTGAAGCCATCGCTTCCTCCT 594.GTCTGAAGCCATCGCTTCCTCCTGAAAAT 595. MSP2193 RUNX1 2 21GGTTTTCGCTCCGAAGGTAAA 596. GGTTTTCGCTCCGAAGGTAAAAGAAAT 597. MSP2195RUNX1 3 21 GGGACTCCCCAAGCCCTATTA 598. GGGACTCCCCAAGCCCTATTAAAAAAT 599.MSP2235 RUNX1 4 22 GCAGCTTGTTTCACCTCGGTGC 600.GCAGCTTGTTTCACCTCGGTGCAGAGAT 601. MSP2194 RUNX1 5 22GACCTGTCTTGGTTTTCGCTCC 602. GACCTGTCTTGGTTTTCGCTCCGAAGGT 603. MSP2216RUNX1 6 23 GCTTCCATCTGATTAGTAAGTAA 604. GCTTCCATCTGATTAGTAAGTAATCCAAT605. MSP2214 RUNX1 7 22 GTGCAGAGATGCCTCGGTGCCT 606.GTGCAGAGATGCCTCGGTGCCTGCCAGT 607. MSP2211 RUNX1 8 21GAGGGTGCATTTTCAGGAGGA 608. GAGGGTGCATTTTCAGGAGGAAGCGAT 609. MSP2217RUNX1 9 23 GTTTCACCTCGGTGCAGAGATGC 610. GTTTCACCTCGGTGCAGAGATGCCTCGGT611. MSP2176 RUNX1 22 GCGATGGCTTCAGACAGCATAT 612.GCGATGGCTTCAGACAGCATATTTGAGT 613. 10 MSP2177 RUNX1 22GCTCCGAAGGTAAAAGAAATCA 614. GCTCCGAAGGTAAAAGAAATCATTGAGT 615. 11 MSP2334RUNX1 22 GAGGCATATGATTACAAGTCTA 616. GAGGCATATGATTACAAGTCTATTGGAT 617.12 MSP2175** RUNX1 21 GAAAGAGAGATGTAGGGCTAG 618.GAAAGAGAGATGTAGGGCTAGAGGGGT 619. 13 MSP2178** RUNX1 23GTACTCACCTCTCATGAAGCACT 620. GTACTCACCTCTCATGAAGCACTGTGGGT 621. 14MSP2210 RUNX1 21 GAGGTGAGTACATGCTGGTCT 622. GAGGTGAGTACATGCTGGTCTTGTAAT623. 15 MSP2213 RUNX1 22 GAGAGGAATTCAAACTGAGGCA 624.GAGAGGAATTCAAACTGAGGCATATGAT 625. 16 MSP2212 RUNX1 21GAGGCTGAAACAGTGACCTGT 626. GAGGCTGAAACAGTGACCTGTCTTGGT 627. 17 VEGFAMSP2196 VEGFA 1 21 GTACATGAAGCAACTCCAGTC 628.GTACATGAAGCAACTCCAGTCCCAAAT 629. MSP2198 VEGFA 2 21GACGGGTGGGGAGAGGGACAC 630. GACGGGTGGGGAGAGGGACACACAGAT 631. MSP2197VEGFA 3 22 GTCCCAAATATGTAGCTGTTTG 632. GTCCCAAATATGTAGCTGTTTGGGAGGT 633.MSP2219 VEGFA 4 21 GGCCAGGGGTCACTCCAGGAT 634.GGCCAGGGGTCACTCCAGGATTCCAAT 635. MSP2220 VEGFA 5 22GCCAGAGCCGGGGTGTGCAGAC 636. GCCAGAGCCGGGGTGTGCAGACGGCAGT 637. MSP2181VEGFA 6 22 GAGGACGTGTGTGTCTGTGTGG 638. GAGGACGTGTGTGTCTGTGTGGGTGAGT 639.MSP2336 VEGFA 7 22 GGGAGAAGGCCAGGGGTCACTC 640.GGGAGAAGGCCAGGGGTCACTCCAGGAT 641. MSP2179** VEGFA 8 21GGGTGAGTGAGTGTGTGCGTG 642. GGGTGAGTGAGTGTGTGCGTGTGGGGT 643. MSP2180VEGFA 9 22 GAGTGAGGACGTGTGTGTCTGT 644. GAGTGAGGACGTGTGTGTCTGTGTGGGT 645.MSP2182 VEGFA 22 GCGTTGGAGCGGGGAGAAGGCC 646.GCGTTGGAGCGGGGAGAAGGCCAGGGGT 647. 10 MSP2218 VEGFA 21GCTCCATTCACCCAGCTTCCC 648. GCTCCATTCACCCAGCTTCCCTGTGGT 649. 11 *Used inFIGS. 1C and 1E, FIG. 32 **Used for GUIDE-seq experiments in FIG. 3,FIGS. 36A-B

Example 3. Engineering the PAM Specificity of Staphylococcus aureus Cas9

Site-specific DNA cleavage by CRISPR-Cas9 nucleases is primarily guidedby RNA-DNA interactions, but also requires Cas9-mediated recognition ofa protospacer adjacent motif (PAM). Although the commonly usedStreptococcus pyogenes Cas9 specifies only two nucleotides within itsNGG PAM, other Cas9 orthologues with desirable properties recognizelonger PAMs. While potentially advantageous from the perspective ofspecificity, extended PAM sequences can limit the targeting range ofCas9 orthologues for genome editing applications. One possible strategyto broaden the range of sequences targetable by such Cas9 orthologuesmight be to evolve variants with relaxed specificity for certainpositions within the PAM. Here we used molecular evolution to modify theNNGRRT (SEQ ID NO:46) PAM specificity of Staphylococcus aureus Cas9(SaCas9), a smaller size orthologue that is useful for applicationsrequiring viral delivery. One variant we identified, referred to as KKHSaCas9, shows robust genome editing activities at endogenous humantarget sites with NNNRRT PAMs. Importantly, using the GUIDE-seq method,we showed that both wild-type and KKH SaCas9 induce comparable numbersof off-target effects in human cells. KKH SaCas9 increased the targetingrange of SaCas9 by nearly two- to four-fold, enabling targeting ofsequences that cannot be altered with the wild-type nuclease. Moregenerally, these results demonstrate the feasibility of relaxing PAMspecificity to broaden the targeting range of Cas9 orthologues. Ourmolecular evolution strategy does not require structural information ora priori knowledge of specific residues that contact the PAM, andtherefore should be applicable to a wide range of Cas9 orthologues.

Results

We devised an unbiased genetic approach for engineering Cas9 variantswith relaxed PAM recognition specificities that does not requirestructural information. We tested this strategy using SaCas9, for whichno structural data was available at the time we initiated these studies.In an initial step, we sought to conservatively estimate thePAM-interacting domain for SaCas9 by sequence comparisons with thestructurally well-characterized SpCas9 (Jiang et al., Science 348,1477-1481 (2015); Anders et al., Nature 513, 569-573 (2014); Jinek etal., Science (2014); Nishimasu et al., Cell (2014)). Although SpCas9 andSaCas9 differ substantially at the primary sequence level (FIG. 21A,FIG. 29), alignment of both with 10 additional orthologues enabled us toconservatively define a predicted PAM-interacting domain for SaCas9 (SeeMethods for Example 3; FIGS. 29 and 30).

Because the guanine at the third position in the SaCas9 PAM is the moststrictly specified base (Ran et al., Nature 520, 186-191 (2015)), werandomly mutagenized the predicted PI domain and used our previouslydescribed bacterial cell-based method (see Examples 1-2) to attempt toselect for mutants capable of cleaving sites with each of the threeother possible nucleotides at the 3^(rd) PAM position (i.e.,NN[A/C/T]RRT PAMs (NNHRRT (SEQ ID NO:44)); FIG. 31A). All but one of thesurviving variants from the selections against sites containing NNARRT(SEQ ID NO:43) and NNCRRT (SEQ ID NO:47) PAMs harbored an R1015Hmutation, whereas we did not obtain any variants from the selectionswith NNTRRT (SEQ ID NO:48) PAMs. These results strongly suggested thatR1015 might participate in recognition of the guanine at the thirdposition of the SpCas9 PAM. Indeed, in our alignments we found thatR1015 of SaCas9 is in the vicinity of SpCas9 R1335 (FIG. 30), a residuepreviously implicated in recognition of the third base position of thePAM ((See Examples 1-2; Anders, C., Niewoehner, O., Duerst, A. & Jinek,M. Structural basis of PAM-dependent target DNA recognition by the Cas9endonuclease. Nature 513, 569-573 (2014)). Consistent with this, wefound that mutation of R1015 to an alanine or glutamine substantiallydecreased SaCas9 activity on a target site containing an NNGRRT (SEQ IDNO:46) PAM (FIG. 21B) when tested in our bacterial selection system(FIG. 31B). Alanine or glutamine substitutions of other positivelycharged residues in the vicinity of R1015 did not have as strong of aneffect on SaCas9 activity (FIG. 21B, FIG. 30).

Our bacterial-based selection results also suggested that the R1015Hmutation might at least partially relax the specificity of SaCas9 at thethird position of the PAM. However, we found that the R1015H singlemutant had suboptimal activity in our previously described humancell-based EGFP disruption assay (Fu et al., Nat Biotechnol 31, 822-826(2013); Reyon et al., Nat Biotechnol 30, 460-465 (2012)) when testedagainst sites with any nucleotide at the 3^(rd) position of NNNRRT PAMs(FIG. 21C). Because this suggested that additional mutations might berequired to increase or optimize the activity of the R1015H mutant inhuman cells, we randomly mutagenized a region encompassing the predictedPI domain of an SaCas9 that also harbored a R1015Q mutation. We thenselected for variants from this library that could cleave target siteswith each of the three different NNHRRT (SEQ ID NO:44) PAMs using ourbacterial selection system. We used R1015Q because, unlike R1015H, thismutant did not show activity in bacteria (FIG. 21B). Although nosurviving clones were again observed when selecting against NNTRRT (SEQID NO:48) PAMs, selections with the R1015Q variant against NNARRT (SEQID NO:43) or NNCRRT (SEQ ID NO:47) yielded mutations at E782, K929,N968, and, surprisingly, mutation of the Q at 1015 to H.

Combined with the selection results from wild-type SaCas9, the mostfrequent missense mutations identified across all selections were E782K,K929R, N968K, and R1015H (FIG. 21D), suggesting that a combination ofthese mutations might permit efficient cleavage of sites that contain anA or C at the third position of the SaCas9 PAM. We therefore testedSaCas9 variants containing different combinations of these mutationsusing the human cell-based EGFP disruption assay with sgRNAs targeted tosites harboring each of the 4 bases at the third position of the PAM(i.e., on NNNRRT PAMs) (FIG. 21E, FIG. 32). We found that the variantswith the triple mutant combinations E782K/N968K/R1015H andE782K/K929R/R1015H were highly active at sites with NNNRRT PAMs (FIG.21E, FIG. 32), whereas the quadruple mutant variant containing all fourmutations (E782K/K929R/N968K/R1015H) had generally lower activities onthese sites (FIG. 32). We chose the E782K/N968K/R1015H (hereafterreferred to as the KKH variant) for further characterization, andverified using our human cell-based EGFP disruption assay that all threesubstitutions comprising the KKH variant were required for activity(FIG. 21E).

To more comprehensively define the PAM specificities of KKH andwild-type SaCas9, we used our previously described bacterial cell-basedsite-depletion assay (See Examples 1-2) (FIG. 33). This method yieldsCas9 PAM specificity profiles by identifying the relative cleavage (andtherefore depletion in bacterial cells) of DNA plasmids bearingrandomized PAM sequences, quantified as a post-selection PAM depletionvalue (PPDV). We performed site-depletion experiments with bothwild-type and KKH SaCas9 using libraries with two different spacersequences each with 8 randomized bases in place of the PAM (FIG. 33).Control experiments using catalytically inactive SaCas9 showed littledepletion of any PAM sequence (FIG. 34A), enabling us to establish athreshold for statistically significant depletion as a PPDV of 0.794(FIG. 34B). Previous experiments have shown that PAMs with PPDVs of <0.2in our bacterial site-depletion assay can be efficiently cleaved in ourhuman cell-based EGFP disruption assay (See Examples 1-2). Withwild-type SaCas9, the most depleted PAMs (based on mean PPDVs obtainedfrom the two libraries) were, as expected, the four NNGRRT (SEQ IDNO:46) (PAMs (FIG. 21F and FIG. 34C). Interestingly, other PAMs withmean PPDVs <0.1 included those of the form NNGRRN (SEQ ID NO:49) (FIG.34), suggesting that for some spacer sequences the last position of thePAM may not be fully specified as a T in our bacterial-based assay(although a previous report demonstrated by an in vitro PAM depletionassay, ChIP-seq, and targeting of endogenous human sites that a thymineat the sixth position of the PAM was highly preferred (Ran, F. A. et al.In vivo genome editing using Staphylococcus aureus Cas9. Nature 520,186-191 (2015))). By contrast, with the KKH variant, PAMs with meanPPDVs of <0.2 included not only the NNGRRT (SEQ ID NO:46) PAMs but alsoall four NNARRT (SEQ ID NO:43), all four NNCRRT (SEQ ID NO:47), andthree of the four NNTRRT (SEQ ID NO:48) PAMs (FIG. 21F, FIGS. 34C and34E). These results suggested that KKH SaCas9 appears to have abroadened PAM targeting range relative to its wild-type counterpart.

To assess the robustness of the KKH SaCas9 variant in human cells, wetested its activity on 55 different endogenous gene target sitescontaining a variety of NNNRRT PAMs (FIG. 22A). The KKH variant showedefficient activity with a mean mutagenesis frequency of 24.7% across allsites, with 80% of sites (44 of 55 sites) showing greater than 5%disruption. Analysis of KKH SaCas9 activity across all 55 sites revealedordered preferences for the 3^(rd) position of the PAM (NN[G>A=C>T]RRT;FIG. 22B) as well as the 4^(th)/5th positions of the PAM(NNN[AG>GG>GA>AA]T; FIG. 22C). Consistent with this, we observeddifferences among the 16 possible combinations of the3^(rd)/4^(th)/5^(th) positions of an NNNRRT PAM (FIG. 35A). KKH SaCas9functioned efficiently on spacer lengths ranging from 21-23 nucleotides(FIG. 22D), spacer sequences with variable GC content (FIG. 35B), andPAMs with variable GC content (FIG. 35C). Sequence logos derived fromsites cleaved with low, medium, and high efficiencies (0-10%, 10-30%,and >30% mean mutagenesis frequencies, respectively) revealed littlesequence preference across the entire target site other than at the4^(th) and 5^(th) positions of the NNNRRT PAM, and perhaps a slightpreference for guanine at the 2^(nd) PAM position on sites cleaved withhigh efficiencies (FIG. 35D).

To demonstrate that the KKH variant enables modification of PAMs thatcannot be targeted by wild-type SaCas9, we performed direct comparisonsof these nucleases in human cells on sites bearing various NNNRRT PAMs.Assessment of 16 sites using our EGFP disruption assay and 16 endogenoushuman gene targets (FIGS. 22E and 22F, respectively) showed that KKHSaCas9 robustly modified target sites bearing NNNRRT PAMs whereaswild-type SaCas9 efficiently targeted only sites with NNGRRT (SEQ IDNO:46) PAMs. For all 24 sites with NNHRRT (SEQ ID NO:44) PAMs, the KKHvariant induced substantially higher rates of mutagenesis than wild-typeSaCas9; on the eight sites with NNGRRT (SEQ ID NO:46) PAMs, KKH SaCas9induced comparable or slightly lower levels of mutagenesis compared withwild-type (FIGS. 22E and 22F). These results collectively demonstratethat the KKH variant can cleave sites with NNNRRT PAMs, thereby enablingtargeting of sites with NNHRRT (SEQ ID NO:44) PAMs that currently cannotbe efficiently altered by wild-type SaCas9 in human cells.

To assess the impact of the KKH mutations on the genome-wide specificityof SaCas9, we used the GUIDE-seq (Genome-wide Unbiased Identification ofDSBs Enabled by sequencing) method (Tsai, S. Q. et al. GUIDE-seq enablesgenome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.Nat Biotechnol 33, 187-197 (2015)) to directly compare the off-targetprofiles of wild-type and KKH SaCas9 with the same sgRNAs. When testedwith sgRNAs targeted to six endogenous human gene sites containingNNGRRT (SEQ ID NO:46) PAMs, we observed that wild-type and KKH SaCas9induced nearly identical GUIDE-seq tag integration rates and on-targetcleavage frequencies for all six sites (FIGS. 36A and 36B,respectively). Furthermore, wild-type and KKH SaCas9 induce mutations atsimilar numbers of off-target sites with each of the six sgRNAs (FIGS.23A and 23B). Off-target sites for the KKH variant generally adhered tothe NNNRRT PAM motif, and off-target sites for wild-type SaCas9 adheredto an NNGRR[T>G] motif (FIG. 22B). With one of the sgRNAs, which inducedthe highest number of off-target sites among the six sgRNAs tested, weobserved a similar number of off-target sites with wild-type and KKHSaCas9. However, the off-target sites were only partially overlappingbetween wild-type and KKH SaCas9, as might be expected given theirdifferent PAM specificities (FIGS. 23B and 23C). Although we would notadvocate the use of the KKH variant for targeting sites with NNGRRT (SEQID NO:46) PAMs (because wild-type SaCas9 can show higher on-targetactivities than KKH for these sites), these results suggest that KKHSaCas9 only cleaves off-target sites with the expected PAMs andgenerally induces numbers of off-target sites comparable to thoseobserved with wild-type SaCas9.

To further examine the genome-wide specificity of KKH SaCas9, we testedfive additional sgRNAs targeted to sites containing NNHRRT (SEQ IDNO:44) PAMs (FIGS. 23D and 23E). Off-target sites detected by GUIDE-seqwere generally low in number (comparable to the numbers observed withwild-type SpCas9 and SpCas9 variants in previously published experiments(See Examples 1-2 (Tsai, S. Q. et al. GUIDE-seq enables genome-wideprofiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol33, 187-197 (2015)), displayed potential DNA- and RNA-bulged off-targets(Lin, Y. et al. CRISPR/Cas9 systems have off-target activity withinsertions or deletions between target DNA and guide RNA sequences.Nucleic Acids Res 42, 7473-7485 (2014)), and contained expected PAMsequences. Taken together, our experiments demonstrate that thegenome-wide specificities of wild-type and KKH SaCas9 are similar andgenerally show low numbers of off-target mutations in human cells asjudged by GUIDE-seq.

Although wild-type SaCas9 remains the most optimal choice for targetingNNGRRT (SEQ ID NO:46) PAMs, the KKH SaCas9 variant we describe here canrobustly target sites with NNARRT (SEQ ID NO:43) and NNCRRT (SEQ IDNO:47) PAMs and has a reasonable success rate for sites with NNTRRT (SEQID NO:48) PAMs. Thus, we conservatively estimate that the KKH variantincreases the targeting range of SaCas9 by nearly two- to four-fold inrandom DNA sequence, thereby improving the prospects for more broadlyutilizing SaCas9 in a variety of different applications that requirehighly precise targeting. Using GUIDE-seq, we demonstrated that KKHSaCas9 induces similar numbers of off-target mutations as wild-typeSaCas9 when targeted to the same sites that contain NNGRRT (SEQ IDNO:46) PAMs. Also, KKH SaCas9 induces only a small number of off-targetmutations when targeted to sites bearing NNHRRT (SEQ ID NO:44) PAMs.Although KKH SaCas9 recognizes a modified PAM sequence relative towild-type SaCas9, our findings are not entirely surprising given thatthe total combined length of the protospacer and PAM is still longenough with the KKH variant (24 to 26 bps) to be reasonably orthogonalto the human genome. Furthermore, it is possible that modifying PAMrecognition can improve specificity by altering the energetics ofCas9/sgRNA interaction with its target site (similar to the previouslyproposed mechanisms for improved specificities of truncated sgRNAs (Fu,Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. ImprovingCRISPR-Cas nuclease specificity using truncated guide RNAs. NatBiotechnol 32, 279-284 (2014)) or the D1135E SpCas9 mutant (See Examples1-2)).

Example 4. Improving the Activity of the SpCas9-VQR Variant

Because the SpCas9-VQR variant has a preference for NGAN PAMs of:NGAG>NGAA=NGAT>NGAC, we sought to select for derivative variants thathad improved activity against NGAH PAMs (where H=A, C, or T). Selectionswith the R1335Q library (with PI domain randomly mutagenized) againstcells that contain target sites with either an NGAA, NGAT, or NGAC PAMenabled us to sequence additional clones that contained mutations thatconvey an altered PAM specificity. The sequences of these clonesrevealed additional mutations that might be important for altering PAMspecificity towards NGAA, NGAT, or NGAC PAMs.

Based on the results of these selections, the VQR variant and 24 otherderivative variants were tested against NGAG, NGAA, NGAT, and NGAC PAMsites in bacteria. A number of these derivative variants survived betterthan the VQR variant on NGAH PAM sites, most of which contained theG1218R mutation (Table 7 and FIG. 24).

TABLE 7 Table of variants and their corresponding amino acid changes.variant D1135 G1218 E1219 R1335 T1337 A1 VRQ V R — Q — A2 NRQ N R — Q —A3 YRQ Y R — Q — A4 VRQL V R — Q L A5 VRQM V R — Q M A6 VRQR V R — Q RA7 VRQE V R — Q E A8 VRQQ V R — Q Q A9 NRQL N R — Q L A10 NRQM N R — Q MA11 NRQR N R — Q R A12 NRQE N R — Q E B1 NRQQ N R — Q Q B2 YRQL Y R — QL B3 YRQM Y R — Q M B4 YRQR Y R — Q R B5 YRQE Y R — Q E B6 YRQQ Y R — QQ B7 VRVQE V R V Q E B8 NRVQE N R V Q E B9 YRVQE Y R V Q E B10 VVQE V —V Q E B11 NVQE N — V Q E B12 YVQE Y — V Q E C1 VQR V — — Q R

Given that the results from the bacterial screen demonstrated that someof these additional mutations improved activity against NGAH PAM sites,we tested some of the best candidates in human cells in the EGFPdisruption assay. What we observed is that a number of these variantsoutperformed the VQR variant at targeting NGAH sites, including theVRQR, NRQR, and YRQR variants (Table 8 and FIG. 25). The main differencebetween these clones and the VQR variant is that they include a G1218Rmutation.

TABLE 8 Table of SpCas9-VQR derivatives and their corresponding aminoacid changes variant D1135 G1218 R1335 T1337 VQR V — Q R YRQ Y R Q —VRQR V R Q R VRQQ V R Q Q NRQR N R Q R NRQQ N R Q Q YRQR Y R Q R YRQQ YR Q Q

Because the VRQR variant appeared to be the most robust of those tested,we compared its activity to that of the VQR against 9 differentendogenous sites in human cells (2 sites for each NGAA, NGAC, NGAT, andNGAG PAMs, and 1 site for an NGCG PAM). This data reveals that the VRQRvariant outperforms the VQR variant at all sites tested in human cells(FIG. 26).

After demonstrating that VRQR variant has improved activity relative tothe VQR variant, we sought to determine whether adding additionalsubstitutions could further improve activity. Because we observedadditional mutations in the selections that were in close proximity tothe PAM interacting pocket of SpCas9, a subset of these mutations wereadded to the VQR and VRQR variants and screened in bacteria againstsites containing NGAG, NGAA, NGAT, and NGAC PAMs (Table 9 and FIG. 27).A number of derivative variants appears to have higher activity againstNGAT and NGAC PAM sites, so we proceeded to test these variants in humancells. We tested in the human cell EGFP disruption assay additionalvariants that contained added mutations to either the VQR or VRQRbackground. These experiments again revealed that the VRQR has morerobust activity against NGAH PAMs than the VQR variant, and thatadditional mutations to the VRQR backbone are beneficial.

TABLE 9 Table of variants and their corresponding amino acid changesvariant mutations 1 VQR + L1111H L1111H/D1135V/R1335Q/T1337R 2 VRQR+ L1111H L1111H/D1135V/G1218R/R1335Q/T1337R 3 VQR + E1219KD1135V/E1219K/R1335Q/T1337R 4 VQR + E1219V D1135V/E1219V/R1335Q/T1337R 5VQR + N1317K D1135V/N1317K/R1335Q/T1337R 6 VRQR + N1317KD1135V/G1218R/N1317K/R1335Q/T1337R 7 VQR + G1104KG1104R/D1135V/R1335Q/T1337R 8 VRQR + G1104KG1104R/D1135V/G1218R/R1335Q/T1337R 9 VQR + S1109TS1109T/D1135V/R1335Q/T1337R 10 VRQR + S1109TS1109T/D1135V/G1218R/R1335Q/T1337R 11 NQR + S1136ND1135N/S1136N/R1335Q/T1337R 12 NRQR + S1136ND1135N/S1136N/G1218R/R1335Q/T1337R 13 VQR D1135V/R1335Q/T1337R 14 VRQRD1135V/G1218R/R1335Q/T1337RTaken together, these results suggest that including additionalmutations in the SpCas9-VQR variant can improve activity against sitesthat contain NGAN PAMs, specifically sites that contain NGAH PAMs.

REFERENCES

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OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-30. (canceled)
 31. An isolated variant Streptococcus pyogenes Cas9(SpCas9) protein, comprising an amino acid sequence that has at least90% sequence identity to SEQ ID NO: 1, with a mutation at G1218 andoptionally at one or more of the following positions: G1104, S1109,L111, E1219, D1135, N1317, R1335, T1337, wherein the isolated variantSpCas9 protein can interact with a guide RNA and a target DNA.
 32. Theisolated variant SpCas9 protein of claim 31, wherein the mutation atG1218 is G1218R.
 33. The isolated variant SpCas9 protein of claim 31,wherein the mutations are: (i) D1135V/G1218R//R1335Q (VRQ variant); (ii)D1135N/G1218R/R1335Q (NRQ variant); (iii) G1218R/D1135Y/R1335Q (YRQvariant); (iv) D1135V/G1218R/R1335Q/T1337L (VRQL variant); (v)D1135V/G1218R/R1335Q/T1337M (VRQM variant); (vi)D1135V/G1218R/R1335Q/T1337E (VRQE variant); (vii)D1135V/G1218R/R1335Q/T1337Q (VRQQ variant); (viii)D1135N/G1218R/R1335Q/T1337L (NRQL variant); (ix)D1135N/G1218R/R1335Q/T1337M (NRQM variant); (x)D1135N/G1218R/R1335Q/T1337E (NRQE variant); (xi)D1135N/G1218R/R1335Q/T1337Q (NRQQ variant); (xii)D1135Y/G1218R/R1335Q/T1337L (YRQL variant); (xiii)D1135Y/G1218R/R1335Q/T1337M (YRQM variant); (xiv)D1135V/G1218R/R1335Q/T1337L (YRQQ variant); (xv)D1135V/G1218R/E1219V/R1335Q/T1337E (VRVQE variant); (xvi)D1135N/G1218R/E1219V/R1335Q/T1337E (NRVQE variant); (xvii)D1135Y/G1218R/E1219V/R1335Q/T1337E (YRVQE variant); or (xviii)D1135N/G1218R/R1335Q/T1337R (NRQR variant).
 34. The isolated variantSpCas9 protein of claim 31, wherein the mutations are: (i)L1111H/D1135V/G1218R/R1335Q/T1337R (LVRQR variant); (ii)D1135V/G1218R/N1317K/R1335Q/T1337R (VRKQR variant); (iii)G1104K/D1135V/G1218R/R1335Q/T1337R (KVRQR variant); (iv)S1109T/D1135V/G1218R/R1335Q/T1337R (TVRQR variant); or (v)D1135N/S1136N/G1218R/R1335Q/T1337R (NNRQR variant).
 35. The isolatedvariant SpCas9 protein of claim 31, further comprising one or moremutations that decrease nuclease activity selected from the groupconsisting of mutations at D10, E762, D839, H983, and D986; and one moremutations selected from the group consisting of mutations at H840 andN863.
 36. The isolated variant SpCas9 protein of claim 35, wherein themutations are: (i) D10A or D10N, and (ii) H840A, H840N, or H840Y. 37.The isolated variant SpCas9 protein of claim 31, wherein the isolatedvariant SpCas9 protein comprises mutations at G1218, D1135, R1335 andT1337.
 38. The isolated variant SpCas9 protein of claim 37, wherein theisolated variant SpCas9 protein comprises mutations selected from thegroup consisting of: G1218R, D1135Y, D1135N, D1135V, R1335Q, T1337R,T1337E, T1337L, T1337M and T1337Q.
 39. The isolated variant SpCas9protein of claim 31, comprising an amino acid sequence that has at least95% sequence identity to the amino acid sequence of SEQ ID NO:1, with amutation at G1218 and optionally at one or more of the followingpositions: G1104, S1109, L1111, D1135, S1136, N1317, R1335, T1337.
 40. Afusion protein comprising the isolated variant SpCas9 protein of claim31, fused to a heterologous functional domain, with an optionalintervening linker, wherein the linker does not interfere with activityof the fusion protein.
 41. The fusion protein of claim 40, wherein theheterologous functional domain is a transcriptional activation domain.42. The fusion protein of claim 41, wherein the transcriptionalactivation domain is from VP64 or NF-κB p65.
 43. The fusion protein ofclaim 40, wherein the heterologous functional domain is atranscriptional silencer or transcriptional repression domain.
 44. Thefusion protein of claim 43, wherein the transcriptional repressiondomain is a Krueppel-associated box (KRAB) domain, ERF repressor domain(ERD), or mSin3A interaction domain (SID).
 45. The fusion protein ofclaim 43, wherein the transcriptional silencer is HeterochromatinProtein 1 (HP1).
 46. The fusion protein of claim 40, wherein theheterologous functional domain is an enzyme that modifies themethylation state of DNA.
 47. The fusion protein of claim 46, whereinthe enzyme that modifies the methylation state of DNA is a DNAmethyltransferase (DNMT) or a ten-eleven translocation (TET) protein.48. The fusion protein of claim 47, wherein the TET protein isten-eleven translocation 1 (TET1).
 49. The fusion protein of claim 40,wherein the heterologous functional domain is an enzyme that modifies ahistone subunit.
 50. The fusion protein of claim 49, wherein the enzymethat modifies a histone subunit is a histone acetyltransferase (HAT),histone deacetylase (HDAC), histone methyltransferase (HMT), or histonedemethylase.
 51. The fusion protein of claim 40, wherein theheterologous functional domain is a biological tether.
 52. The fusionprotein of claim 51, wherein the biological tether is MS2, Csy4 orlambda N protein.
 53. The fusion protein of claim 40, wherein theheterologous functional domain is FokI.
 54. An isolated nucleic acidencoding the isolated variant SpCas9 protein of claim
 31. 55. A vectorcomprising the isolated nucleic acid of claim 54, optionally operablylinked to one or more regulatory domains.
 56. The vector of claim 55,wherein the vector is a prokaryotic vector.
 57. The vector of claim 56,wherein the prokaryotic vector is a plasmid, a shuttle vector, or aninsect vector.
 58. The vector of claim 55, wherein the vector is anexpression vector.
 59. A host cell expressing the isolated variantSpCas9 protein of claim
 31. 60. A method of altering the genome of acell, the method comprising expressing in the cell, or contacting thecell with, (i) an isolated variant Streptococcus pyogenes Cas9 (SpCas9)protein, comprising an amino acid sequence that has at least 90%sequence identity to SEQ ID NO: 1, with a mutation at G1218 andoptionally at one or more of the following positions: G1104, S1109,L111, E1219, D1135, N1317, R1335, T1337, wherein the isolated variantSpCas9 protein can interact with a guide RNA and a target DNA, or (ii) afusion protein comprising an isolated variant Streptococcus pyogenesCas9 (SpCas9) protein fused to a heterologous functional domain, with anoptional intervening linker, wherein the linker does not interfere withactivity of the fusion protein, wherein the isolated variant SpCas9protein comprises an amino acid sequence that has at least 90% sequenceidentity to SEQ ID NO: 1, with a mutation at G1218 and optionally at oneor more of the following positions: G1104, S1109, L111, E1219, D1135,N1317, R1335, T1337, and wherein the isolated variant SpCas9 protein caninteract with a guide RNA and a target DNA, and a guide RNA having aregion complementary to a selected portion of the genome of the cell.61. The method of claim 30, wherein the cell is a stem cell.
 62. Themethod of claim 30, wherein the cell is an isolated embryonic stem cell,an isolated mesenchymal stem cell, or an induced pluripotent stem cell.63. The method of claim 30, wherein the cell is in a living animal or isin an embryo.
 64. A method of altering a double stranded DNA (dsDNA)molecule, the method comprising contacting the dsDNA molecule with (i)an isolated variant Streptococcus pyogenes Cas9 (SpCas9) protein,comprising an amino acid sequence that has at least 90% sequenceidentity to SEQ ID NO: 1, with a mutation at G1218 and optionally at oneor more of the following positions: G1104, S1109, L111, E1219, D1135,N1317, R1335, T1337, wherein the isolated variant SpCas9 protein caninteract with a guide RNA and a target DNA, or (ii) a fusion proteincomprising an isolated variant Streptococcus pyogenes Cas9 (SpCas9)protein fused to a heterologous functional domain, with an optionalintervening linker, wherein the linker does not interfere with activityof the fusion protein, wherein the isolated variant SpCas9 proteincomprises an amino acid sequence that has at least 90% sequence identityto SEQ ID NO: 1, with a mutation at G1218 and optionally at one or moreof the following positions: G1104, S1109, L111, E1219, D1135, N1317,R1335, T1337, and wherein the isolated variant SpCas9 protein caninteract with a guide RNA and a target DNA, and a guide RNA having aregion complementary to a selected portion of the dsDNA molecule. 65.The method of claim 34, wherein the dsDNA molecule is in vitro.